FIELD OF THE INVENTION
[0001] The invention relates to orally available dipeptides capable of modulating intracellular
gap junctional communication. The invention also relates to methods of using the peptides
to modulate such communication, to the use of the peptides for the manufacture ofmedicaments
for the prevention and/or treatment of conditions associated with said communication
and to pharmaceutical compositions comprising said dipeptides.
BACKGROUND
[0003] There have been efforts to understand the structure and function of gap junctions.
For instance, there have been reports that such junctions are a type of complex formed
between adjacent cells. Most gap junctions are thought to consist of aggregated channels
that directly link the interiors (cytoplasms) of neighboring cells. In adult mammals,
gap junctions are found in most cell types with the exception of circulating blood
elements.
[0004] More specifically, there is acknowledgement that gap junctions are specialized regions
of the cell membrane with clusters of hundreds to thousands of densely packed gap
junction channels (comprising two hemichannels or connexins). Many are thought to
directly connect the cytoplasmic compartments of two neighboring cells. The gap junction
channel can switch between an open and a closed state. In the open state ions and
small molecules are thought to pass through the pore. The conduction of electrical
impulses and intercellular diffusion of signaling molecules take place through the
gap junctions.
[0005] The "cross-talk" between gap junctions has been referred to as gap junctional intracellular
communication (GJIC), which is believed to play an important role in the regulation
of cell metabolism, proliferation, cell-to-cell signaling, and tissue integrity.
[0006] For instance, GJIC is thought to permit rapid equilibration of nutrients, ions, and
fluids between cells. Gap junctions are also thought to serve as electrical synapses
in electrically excitable cells. In many tissues, electrical coupling is thought to
permit more rapid cell-to-cell transmission of action potentials than chemical synapses.
In cardiomyocytes and smooth muscle cells, for instance, this is thought to assist
synchronous contraction.
[0007] There have been reports of other functions mediated by GJIC. For example, GJIC is
thought to enhance the responsiveness of tissues to external stimuli. Second messengers
are generally believed to be small enough to pass from hormonally activated cells
to quiescent cells through junctional channels and activate the latter.
[0008] Additionally, there have been reports that gap junctions may provide intercellular
pathways for chemical and/or electrical developmental signals and assist in defining
the boundaries of developmental compartments. It has been disclosed that GJIC occurs
in specific patterns in embryonic cells and the impairment of GJIC has been related
to developmental anomalies and the teratogenic effects of many chemicals. Further,
GJIC is thought to assist coordination of cell activities.
[0010] Accordingly, there is recognition in the field of a relationship between a malfunction
or absence of gap junctions and an increased risk of arrhythmias. There is thought
to be a further relationship between altered connexin expression/distribution and
chronic heart disease.
[0011] Attempts to analyze peptides that influence GJIC have been made. For instance, a
group of peptides (the antiarrhythmic peptides) have been disclosed with capacity
to increase gap junction conductance in the heart. In particular, a hexapeptide with
a molecular weight of 470D was reported to have been isolated from bovine atria. In
neonatal rat cardiomyocytes, it was reported that the peptide could convert fibrillation
induced by ouabain, calcium, or potassium, to normal rhythm. In addition, the peptide
has been reported to convert arrhythmic movement of isolated rat atria induced by
the combination of potassium and acetylcholine to normal rhythm. This peptide is often
referred to as antiarrhytmic peptide (AAP). See eg.,
Aonuma, S., et al. (1980) Chem Pharm Bull (Tokyo) 28: 3332-3339
[0012] There is increasing understanding that AAP is an important peptide with capacity
to modulate GJIC in the heart.
[0013] For example, in cell culture, AAP has been shown to increase the number of beating
centers, the relative content of spreading cells, and protein synthesis. See
Aonuma, S., et al. (1980) Chem Pharm Bull (Tokyo) 28: 3340-3346. In other studies, the antiarrhythmic effect of AAP observed in
vitro has been confirmed. It has been reported that AAP is effective against CaCl
2-, oubain- and acotinine-induced arrhythmia in mice. See
Ronsberg, M.A., et al. (1986) Med. Sci. 14: 350-351.
[0017] There is increasing understanding that many of the antiarrhythmic peptides positively
impact GJIC, often without affecting action potential duration or shape. Further,
many of such peptides are thought to lack undesirable proarrhythmic side-effects.
Such effects are thought to limit the use of many currently available antiarrhythmic
drugs. Moreover, AAP, as well as certain AAP derivatives, are thought to have some
undesired features, e.g., low stability and a need for high doses before therapeutic
efficacy is achieved.
[0018] Many potential important peptides suffer from a lack of acceptable oral availability.
That is, the peptides are degraded in the gastrointestinal tract, enterocytes, or
both. In such cases, the peptides are often administered to subjects by more painful
and less convenient intravenous routes.
[0019] There have been attempts to improve the oral availability of certain compounds by
enhancing contact with an intestinal peptide transporter protein called PepT1. Much
is known about the PepT1 transporter system. See
Bailey, P.D., et al. (2000) Angew. Chem. Int. Ed. 39:506; and references cited therein.
[0020] It would be desirable to have more effective peptide modulators of GJIC. It would
be especially desirable to have peptide modulators that are orally available.
SUMMARY OF THE INVENTION
[0022] The invention generally relates to peptides that modulate gap junction intercellular
communication (GJIC) as set out in the claims. Preferred peptides are orally available.
The invention has a wide spectrum of useful applications including use in the treatment
or prevention of pathologies associated with impaired GJIC.
[0023] Accordingly, in a first aspect, the present invention provides a peptide for use
in therapy represented by the general formula I:
wherein:
a is 1 and b is 0; or
b is 1 and a is 0;
d is 0-8; and
z is 1-7; and
x is 1, y and q are 1, and p is 0; or
p is i , x and q are 0, and y is 1;
and further wherein,
if R1 is H then d is 0-8; or
if R1 is not H then d is 0;
wherein R1 is the side chain of an amino acid selected from the group consisting of alanine,
arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, and valine;
wherein R2 is selected from the group consisting of NH2, NHR, NR2, NR3+H, OH, SH, RO, RS, RSO, RSO2, COR, CSR, COOH, COOR, CONH2, CONHR, CONR2, OCOR, and SCOR, wherein R = alkyl, alkenyl, aryl, aralkyl, or cycloalkyl;
wherein R3 is H or CH3; and
wherein RX is a hydrophobic group;
or a pharmaceutically acceptable salt thereof.
[0024] Further, the invention concerns a peptide represented by the general formula II:
wherein:
a is 1 and b is 0; or
b is 1 and a is 0;
d is 0-8; and
z is 1-7; and
x is 1, y and q are 1, and p is 0; or
p is 1, x and q are 0, and y is 1;
and further wherein,
if R1 is H then d is 0-8; or
if R1 is not H then d is 0;
wherein R1 is the side chain of an amino acid selected from the group consisting of alanine,
arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, and valine;
wherein R2 is selected from the group consisting of NH2, NHR, NR2, NR3+H, OH, SH, RO, RS, RSO, RSO2, COR, CSR, COOH, COOR, CONH2, CONHR, CONR2, OCOR, and SCOR, wherein R = alkyl, alkenyl, aryl, aralkyl, or cycloalkyl;
wherein R3 is H or CH3:
wherein R4 and R5 are independently selected from the group consisting of H, alkyl, alkenyl, aryl,
aralkyl, halogen, CN, NO2, alkoxy, aryloxy, aralkyloxy, thioalkoxy, thioaryloxy, thioaralkyloxy, +S(CH3)2, SO3H, SO2R, NH2, NHR, NR2, +NR3, OH, SH, COOH, COOR, CONH2, CONHR, CONR2, CH2OH, NCO, NCOR, NHOH, NHNH2, NHNRH, CH2OCOR, CH2OCSR, COR, CSR, CSOR, CF3, and CCl3, and wherein R is alkyl, alkenyl, aryl, aralkyl, or cycloalkyl;
or a pharmaceutically acceptable salt thereof.
[0025] Such peptides may also comprise a peptide bond that is alkylated or otherwise modified
to stabilize the peptide against enzymatic degradation and/or may comprise D-amino
acids.
[0026] More preferred peptides as represented by Formula I and II are orally available to
a mammal, and particularly, to a human subject. Various assays to establish oral availability
are known in the field and include, but are not limited to, the following specific
tests.
Oral availability assays
[0027] One assay ("the Bailey assay") is a predictive assay and is generally followed in
accordance with the invention by other empirical assays, described further below.
In the Bailey assay, candidate peptides are virtually aligned with a substrate template
that is modeled to represent a compound, which binds to PepT1, to identify peptides
likely to bind to PepT1
in vitro and
in vivo. The Bailey assay can be used to facilitate detection of orally available peptides
of the invention.
[0028] In one embodiment, the peptides according to Formula I and II are used in substantial
agreement with this substrate template model (e.g., the peptides show substantially
the same three-dimensional conformation as previously identified substrates which
bind to the hPepT1 with high affinity) and therefore are good candidates for peptides,
which are orally available. Thus, such peptides are typically dipeptides.
[0029] More preferred peptides are those which exhibit at least the following four properties
ascribed to high affinity substrates for PepT1; i.e., from the N- to the C-terminus
of the peptide, the peptide comprises: 1) a strong binding site for an N-terminal
NH
3+ group; 2) a hydrogen bond to the carbonyl group of the first peptide bond; 3) a hydrophobic
pocket preferably featuring strong directional vector and 4) a carboxylate binding
site. Preferably, the first carboxylate binding site is followed by a second carboxylate
binding site according to the model. See, e.g., Bailey, P.D., et al, (2000)
supra.
[0030] One empirical assay to validate the predictive Bailey assay described above, is a
"standard
in vivo oral availability assay". In this assay, a peptide is orally administered to a mammal
and blood samples are taken over time. The concentration of the peptide is determined
at different time intervals using standard quantitation assays such as LC/MS/MS to
calculate an area under the curve (AUC) from a plot of plasma protein concentration
vs. time, using routine methods known in the art. Preferably, different doses of peptides
are administered to a plurality of animals in parallel to identify those peptides
that show a dose-proportional increase in maximum plasma concentration and AUC values.
As a control, the same concentrations of peptide may be administered intravenously
and the area under AUC obtained for oral administration can be compared to the AUC
obtained for intravenous administration. See, e.g.,
Milo Gibal (1991) Biopharmaceutics and Pharmacology, 4th edition (Lea and Sediger). In a preferred embodiment peptides with good oral availability are those, in which
the dose normalised AUC after oral administration represents at least 20% of the dose
normalised AUC after intravenously administration.
[0031] Peptides which show good oral availability according to the standard
in vivo oral availability assay described above, may or may not be in substantial agreement
with the Bailey assay.
[0032] It will often be useful to perform a second empirical assay to further validate orally
available peptides as represented by Formula I or II. In one such assay, the ability
of the peptide to bind to the hPepT1 transporter or a functionally active fragment
thereof is determined. Preferably, peptides according to the invention have good affinity
for an hPepT1 transporter or a biologically active fragment thereof. Preferably, the
K
i of such peptides is less than about 20 mM, more preferably, is less than about 10
mM, and still more preferably, is less than about 5 mM, or less than about 1 mM.
[0033] An hPepT1 binding assay may be performed as an initial screen for peptides which
are then screened for in a standard
in vivo oral availability assay and/or may be performed to confirm the results of a standard
in vivo oral availability assay; however, the standard
in vivo oral availability assay provides the most meaningful test of the peptide oral availability.
Additionally preferred peptides as represented by Formula I above, exhibit a good
half-life according to what is referred to herein as an "
in vitro plasma stability assay" or related phrase. Such peptides may be in substantial agreement
with the foregoing PepT1 substrate template model (Bailey assay). However, for some
peptides there may be little or no agreement with the model. Peptides that show a
good stability in the assay have in one embodiment a half-life of more than about
48 hours, such as more than 24 hours, for example more than 12 hours, such as more
than 6 hours, for example more than 3 hours, such as more than 1 hour, for example
more than 30 minutes. In this embodiment, the peptides of the invention show enhanced
stability in the bloodstream.
[0034] Peptides within the scope of the present invention are in one embodiment represented
herein with free N-terminal and/or C-terminal group. These groups may remain free
for some invention uses. However, in another embodiment, the peptides can feature
blocked C-terminal groups and free N-groups. Alternatively, such peptides may have
blocked N-groups and free C-terminal groups, or blocked N- and C-terminal groups.
The nature of the terminal groups at either side of the molecule is not critical so
long as the peptides are able to bind with satisfactory affinity to hPepT1 such that
the peptides and/or are orally available. As discussed, "satisfactory affinity" for
the hPepT1 transporter can be predicted, in advance, by determining if a subject peptide
is in substantial agreement with the substrate template model for binding to the hPepT1
carrier. See Bailey, P.D., et al. (2000),
supra.
[0035] Additionally, amino acids residues within the dipeptides may be D- or L-amino acids.
In certain aspects, to enhance stability of the compounds, D- amino acids are preferred.
[0036] The peptides according to the invention have a wide variety of important uses and
advantages.
[0037] For instance, such peptides may be used for preventing and/or treating conditions
associated with impaired gap junction function resulting in reduced intercellular
communication or overcoupling of intercellular communication or misregulated cellular
communication and be used for the manufacture of a medicament for preventing an/or
treating conditions associated with impaired gap junction function. In one aspect,
the invention provides a method of administering to a patient having, or at risk of
developing such a condition, a therapeutically effective amount of any of the peptides
described above. Preferably, administration is oral. In one preferred aspect, a patient
is a human being.
[0038] Examples of conditions which can be treated include, but are not limited to, cardiovascular
disease, inflammation of airway epithelium, disorders of alveolar tissue, bladder
incontinence, impaired hearing due to diseases of the cochlea, endothelial lesions,
diabetic retinopathy and diabetic neuropathy, ischemia of the central nervous system
and spinal cord, dental tissue disorders including periodontal disease, kidney diseases,
failures of bone marrow transplantation, wounds, erectile dysfunction, urinary bladder
incontinence, neuropathic paln, subchronic and chronic inflammation, cancer and failures
of bone marrow and stern cell transplantation, conditions which arise during transplantation
of cells and tissues or during medical procedures such as surgery.
[0039] These treatments may employ pharmaceutical compositions comprising any of the peptides
described above and a pharmaceutically acceptable carrier. Preferably, the carrier
is sterile, pyrogen-free and virus-free. Still more preferably, the composition is
orally adminstrable.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040]
Figures 1A and 1B show exemplary synthesis schemes for generating peptides according
to certain aspects of the invention.
Figure 2 shows the effect of Compound 4, Compound 22, Compound 23 and Compound 95
on ALP (Alkaline Phosphatase) in human osteoblasts.
DETAILED DESCRIPTION OF THE INVENTION
[0041] As discussed, the invention relates to orally available peptides that modulate gap
junction intercellular communication (GJIC). The invention has a wide spectrum of
useful applications including use in the treatment or prevention of pathologies associated
with impaired GJIC. Particular invention peptides are represented by Formula I above.
[0042] More specific peptides according to the invention are represented by the following
general formula II as defined above and in the claims.
[0043] As discussed, the peptide can include a free N-terminal, or a free C-terminal, or
both.
[0044] Preferably, the peptide comprises at least one hydrophobic group and at least one
hydrogen bond group. In one aspect of the invention the three dimensional structure
of the peptide the distance between the at least one hydrophobic group and the at
least one hydrogen bonding group Is from about 4 Ångstrøms to about 12 Ångstrøms,
preferably 5 to about 10 Ångstrøms. More preferably, the hydrophobic group is an aromatic
group, including, but not limited to, a benzoyl or benzyl group, or substituted, or
modified forms thereof. Substituted benzoyl or benzyl groups preferably comprise substituents
at the 4- position, More preferably, the substitutents have a radius of 3-11 Ångstrøms
In the sphere taken up by the substitutent. Suitable substituents Include, but are
not limited to: methyl, ethyl, t-butyl, c-hexyl, phenyl, n-butyl, n-hexyl, n-octyl,
ethoxy, t-butoxy, phenoxy, butoxy, benzyloxy, n-hexyloxy, n-octyloxy.
[0045] More specific peptides within the scope of the Invention and having this general
formula are shown in Table 1.
Table 1 |
|
Compound No. |
Compound Name |
|
|
1 |
H-Gly-Lys(4-nitrobenzoyl)-OH |
2 |
H-Gly-Lys(4-fluorobenzoyl)-OH |
3 |
H-Gly-Lys(4-cyanobenzoyl)-OH |
4 |
H-Gly-Lys(4-methoxybenzoyl)-OH |
5 |
H-Gly-Lys(4-chlorobenzoyl)-OH |
6 |
H-Gly-Lys(benzoyl)-OH |
7 |
H-Lys(4-nitrobenzoyl)-Gly-OH |
8 |
H-Lys(4-nitrobenzoyl)-Sar-OH |
9 |
H-Lys(benzoyl)-Sar-OH |
10 |
H-Lys(benzoyl)-Gly-OH |
11 |
H-Lys(4-methoxybenzoyl)-Gly-OH |
12 |
H-Gly-D-Lys(4-methoxybenzoyl)-OH |
13 |
H-Gly-D-Lys(4-nitrobenzoyl)-OH |
14 |
H-Gly-D-Lys(4-fluorobenzoyl)-OH |
15 |
H-Gly-D-Lys(4-cyanobenzoyl)-OH |
16 |
H-Gly-D-Lys(4-nitrobenzoyl)-OH |
17 |
H-Gly-D-Lys(benzoyl)-OH |
18 |
H-Lys(4-fluorobenzoyl)-Gly-OH |
19 |
H-Lys(4-cyanobenzoyl)-Gly-OH |
20 |
H-Lys(4-chlorobenzoyl)-Gly-OH |
21 |
H-D-Lys(4-methoxybenzoyl)-Gly-OH |
22 |
H-D-Lys(4-nitrobenzoyl)-Gly-OH |
23 |
H-D-Lys(benzoyl)-Gly-OH |
24 |
H-D-Lys(4-fuorobenzoyl)-Gly-OH |
25 |
H-D-Lys(4-cyanobenzoyl)-Gly-OH |
26 |
H-D-Lys(4-chlorobenzoyl)-Gly-OH |
27 |
H-Lys(4-cyanobenzoyl)-Sar-OH |
28 |
H-Lys(4-methoxybenzoyl)-Sar-OH |
29 |
H-Lys(4-fuorobenzoyl)-Sar-OH |
30 |
H-Lys(4-chlorobenzoyl)-Sar-OH |
31 |
H-D-Lys(4-cyanobenzoyl)-Sar-OH |
32 |
H-D-Lys(4-methoxybenzoyl)-Sar-OH |
33 |
H-D-Lys(4-fuorobenzoyl)-Sar-OH |
34 |
H-D-Lys(4-chlorobenzoyl)-Sar-OH |
35 |
H-D-Lys(4-nitrobenzoyl)-Sar-OH |
36 |
H-D-Lys(benzoyl)-Sar-OH |
37 |
H-Ala-D-Lys(4-methoxybenzoyl)-OH |
38 |
H-Val-D-Lys(4-methoxybenzoyl)-OH |
39 |
H-Ile-D-Lys(4-methoxybenzoyl)-OH |
40 |
H-Leu-D-Lys(4-methoxybenzoyl)-OH |
41 |
H-Phe-D-Lys(4-methoxybenzoyl)-OH |
42 |
H-Trp-D-Lys(4-methoxybenzoyl)-OH |
43 |
H-His-D-Lys(4-methoxybenzoyl)-OH |
44 |
H-Tyr-D-Lys(4-methoxybenzoyl)-OH |
45 |
H-D-Lys(4-methoxybenzoyl)-Ala-OH |
46 |
H-D-Lys(4-methoxybenzoyl)-Phe-OH |
47 |
H-D-Lys(4-methoxybenzoyl)-Ile-OH |
48 |
H-D-Lys(4-methoxybenzoyl)-Leu-OH |
49 |
H-D-Lys(4-methoxybenzoyl)-Val-OH |
50 |
H-D-Lys(4-methoxybenzoyl)-His-OH |
51 |
H-D-Lys(4-methoxybenzoyl)-Trp-OH |
52 |
H-D-Lys(4-methoxybenzoyl)-Tyr-OH |
53 |
H-D-Lys(4-phenoxybenzoyl)-Gly-OH |
54 |
H-D-Lys(4-t-butylbenzoyl)-Gly-OH |
55 |
H-D-Lys(4-n-butoxybenzoyl)-Gly-OH |
56 |
H-D-Lys(4-methylbenzoyl)-Gly-OH |
57 |
H-D-Lys(4-ethylbenzoyl)-Gly-OH |
58 |
H-D-Lys(4-n-butylbenzoyl)-Gly-OH |
59 |
H-D-Lys(4-n-hexylbenzoyl)-Gly-OH |
60 |
H-D-Lys(4-n-octylbenzoyl)-Gly-OH |
61 |
H-D-Lys(4-pheylbenzoyl)-Gly-OH |
62 |
H-D-Lys(4-benzyloxybenzoyl)-Gly-OH |
63 |
H-D-Lys(4-ethoxybenzoyl)-Gly-OH |
64 |
H-Asn(NH(4-trifluoromethylbenzyl))-Ala-OH |
65 |
H-Asn(NH(4-methoxybenzyl))-Ala-OH |
66 |
H-Asn(NH(4-nitrobenzyl))-Ala-OH |
67 |
H-Asn(NH(benzyl))-Ala-OH |
68 |
H-Asn(NH(4-fluorobenzyl))-Ala-OH |
69 |
H-Asn(NH(4-chlorobenzyl))-Ala-OH |
70 |
H-Asn(NH(4-cyanobenzyl))-Ala-OH |
71 |
H-Asn(NH(4-methylbenzyl))-Ala-OH |
72 |
H-Asn(NH(4-n-butylbenzyl))-Ala-OH |
73 |
H-Asn(NH(4-t-butylbenzyl))-Ala-OH |
74 |
H-Asn(NH(4-n-hexylbenzyl))-Ala-OH |
75 |
H-Asn(NH(4-n-octylbenzyl))-Ala-OH |
76 |
H-Asn(NH(4-phenylbenzyl))-Ala-OH |
77 |
H-Asn(NH(4-phenoxybenzyl))-Ala-OH |
78 |
H-Asn(NH(4-n-butoxybenzyl))-Ala-OH |
79 |
H-Asn(NH(4-trifluoromethylbenzyl))-D-Ala-OH |
80 |
H-Asn(NH(4-methoxybenzyl))-D-Ala-OH |
81 |
H-Asn(NH(4-nitrobenzyl))-D-Ala-OH |
82 |
H-Asn(NH(benzyl))-D-Ala-OH |
83 |
H-Asn(NH(4-fluorobenzyl))-D-Ala-OH |
84 |
H-Asn(NH(4-chlorobenzyl))-D-Ala-OH |
85 |
H-Asn(NH(4-cyanobenzyl))-D-Ala-OH |
86 |
H-Asn(NH(4-methylbenzyl))-D-Ala-OH |
87 |
H-Asn(NH(4-n-butylbenzyl))-D-Ala-OH |
88 |
H-Asn(NH(4-t-butylbenzyl))-D-Ala-OH |
89 |
H-Asn(NH(4-n-hexylbenzyl))-D-Ala-OH |
90 |
H-Asn(NH(4-n-octylbenzyl))-D-Ala-OH |
91 |
H-Asn(NH(4-phenylbenzyl))-D-Ala-OH |
92 |
H-Asn(NH(4-phenoxybenzyl))-D-Ala-OH |
93 |
H-Asn(NH(4-n-butoxybenzyl))-D-Ala-OH |
94 |
H-D-Asn(NH(4-trifluoromethylbenzyl))-Ala-OH |
95 |
H-D-Asn(NH(4-methoxybenzyl))-Ala-OH |
96 |
H-D-Asn(NH(4-nitrobenzyl))-Ala-OH |
97 |
H-Asn(NH(4-methoxybenzyl))-Sar-OH |
98 |
H-Asn(NH(4-methoxybenzyl))-Leu-OH |
99 |
H-Asn(NH(4-methoxybenzyl))-Phe-OH |
100 |
H-Gln(NH(4-methoxybenzyl))-Ala-OH |
101 |
H-Orn(4-methoxybenzoyl)-Gly-OH |
102 |
H-Gly-Asn(NH(4-methoxybenzyl))-OH |
103 |
H-D-Lys(2,4-dinitrobenzoyl)-Gly-OH |
104 |
H-D-Lys(2,4-dimethylbenzoyl)-Gly-OH |
105 |
H-D-Lys(2,5-dimethylbenzoyl)-Gly-OH |
106 |
H-D-Lys(3,5-dimethylbenzoyl)-Gly-OH |
107 |
H-D-Lys(2,4-dichlorobenzoyl)-Gly-OH |
108 |
H-D-Lys(2,5-dichlorobenzoyl)-Gly-OH |
109 |
H-D-Lys(4-fluoro-3-nitrobenzoyl)-Gly-OH |
110 |
H-D-Lys(3-fluoro-4-methylbenzoyl)-Gly-OH |
[0046] More particular peptides according to the invention in one aspect facilitate and/or
maintain or inhibit the intercellular communication mediated by gap junctions. In
one embodiment the peptides may be antiarrhythmic peptides which target the same cells
targeted by AAP, AAP10, HP5, and/or functional analogs thereof, i.e., the peptides
are able to modulate the function of these cells by agonizing or antagonizing the
function of AAP, AAP10, HP5, and/or functional analogs thereof. The invention also
relates to the preparation and use of pharmaceutical compositions for the treatment
of pathologies associated with impaired intercellular gap junctional communication
and methods for using these compositions.
[0047] Further preferred peptides in accord with the invention show good activity in one
or more of the following assays. Although not necessary to identify orally available
peptides as represented by Formulae I and II, above, they can be used to further confirm
and optionally quantify activity of one or a pool of peptides.
[0048] In one embodiment of the invention the peptide is selected from the group consisting
of H-Gly-Lys(4-nitrobenzoyl)-OH (Compound 1); H-Gly-Lys(4-methoxybenzoyl)-OH (Compound
4); H-D-Lys(4-methoxybenzoyl)-Gly-OH (Compound 21); H-
D-Lys(4-nitrobenzoyl)Gly-OH (Compound 22); H-D-Lys(4-t-butylbenzoyl)-Gly-OH (Compound
54); and H-
D-Asn(NH(4-nitrobenzyl)Ala-OH (Compound 96).
[0049] In another embodiment the present peptide is selected from the group consisting of
H-
D-Lys(benzoyl)Gly-OH (Compound 23) and H-
D-Asn(NH(4-methoxybenzyl)Ala-OH (Compound 95).
[0050] In yet another embodiment of the invention the peptide is H-Gly-Lys(4-nitrobenzoyl)-OH
(Compound 1).
[0051] According to the invention in one aspect the peptide is H-Gly-Lys(4-methoxybenzoyl)-OH
(Compound 4).
[0052] Further, in yet another embodiment the peptide is H-D-Lys(4-methoxybenzoyl)-Gly-OH
(Compound 21).
[0053] In yet another aspect the peptide is H-
D-Lys(4-nitrobenzoyl)Gly-OH (Compound 22).
[0054] In a further aspect the peptide is H-D-Lys(4-t-butylbenzoyl)-Gly-OH (Compound 54).
[0055] In still another aspect the peptide is H-
D-Asn(NH(4-nitrobenzyl)Ala-OH (Compound 96).
[0056] Further, in yet another embodiment the peptide peptide is H-
D-Lys(benzoyl)Gly-OH (Compound 23).
[0057] In still another embodiment the peptide is H-
D-Asn(NH(4-methoxybenzyl)Ala-OH (Compound 95).
[0058] Preferred peptides according to Formulae I and II above show good function as a modulator
of gap junctional communication (e.g., as agonists or antagonists). In one aspect
the peptides have the function as an antiarrhythmic drug.
[0059] In one embodiment the preferred agonist peptides of the invention provide an intracellular
conductance (Gj) that is substantially the same as, or is greater than, the Gj of
AAP in what is referred to herein as a "standard cardiomyocyte assay".
[0060] In another embodiment the preferred antagonist peptides provide a Gj that is less
than the Gj of AAP and/or block the ability of AAP to normalize the Gj of an ischemic
cell, i.e., to return the Gj to substantially the same values found in non-ischemic
cells. In one aspect of the invention the present antagonists provide a Gj that is
at least about 40% less than the Gj of AAP, such as at least about 50% less, for example
at least about 60% less, such as at least about 70% less, for example at least about
80% less, such as at least 90% less, for example at least 100% less.
[0061] The inhibitory properties of the present antagonists are not limited to the relative
Gj as described above. In further embodiments and assay types the antagonists may
display a different relative Gj. This may further depend on the comparative peptide/compound.
Heart
[0062] Additionally preferred peptides according to the invention increase the time to an
AV block in a mouse after infusion of CaCl
2, in what is referred to herein as a "standard calcium-induced arrhythimia assay".
Preferably, the peptides provide at least about 40% of the activity of AAP, for example
at least about 50% of the activity of AAP, such as about 60% of the activity of AAP,
for example at least about 70% of the activity of AAP, such as at least about 80%
of the activity of AAP, for example at least about 90% of the activity of AAP, for
example at least about substantially the same activity of AAP, such as about 110%
of the activity of AAP, for example at least about 120% of the activity of AAP, such
as at least about 130% of the activity of AAP, for example at least about 140% of
the activity of AAP, such as about 150% of the activity of AAP, for example at least
about 160% of the activity of AAP, such as at least about 170% of the activity of
AAP, for example at least about 180% of the activity of AAP, preferably at least about
190% of the activity of AAP, more preferably at least about 200 or greater % of the
activity of AAP (i.e., show time lags of approximately the same duration).
[0063] Peptides may additionally show decreases in the incidence of reentry arrhythmias
or in the size of an infarct zone observed in what is referred to herein as a "standard
ventricular reentry assay". Preferably, the peptides provide at least about 40% of
the activity of AAP, for example at least about 50% of the activity of AAP, such as
about 60% of the activity of AAP, for example at least about 70% of the activity of
AAP, such as at least about 80% of the activity of AAP, for example at least about
90% of the activity of AAP, for example at least about substantially the same activity
of AAP, such as about 110% of the activity of AAP, for example at least about 120%
of the activity of AAP, such as at least about 130% of the activity of AAP, for example
at least about 140% of the activity of AAP, such as about 150% of the activity of
AAP, for example at least about 160% of the activity of AAP, such as at least about
170% of the activity of AAP, for example at least about 180% of the activity of AAP,
preferably at least about 190% of the activity of AAP, more preferably at least about
200 or greater % of the activity of AAP (i.e., providing similar decreases in incidence
or infarct zones of similar or smaller size).
Osteoporosis
[0064] There is understanding that GJIC is important in bone formation. Additionally preferred
peptides additionally, or alternatively, increase osteoblast activity in what is referred
to herein as a "standard osteoblast activity assay" which measures either calcium
wave formation and/or alkaline phosphatase activity of osteoblast cells in the presence
of peptides. Preferably, such peptides increased calcium wave activity, manifested
as an increase in the number of cells involved in a wave (as determined by measuring
levels of intracellular Ca
2+ using a calcium sensitive fluorescent dye, such as fura-2 and counting the number
of cells which fluoresce). Alkaline phosphatase activity also can be used to provide
a measure of osteoblast activity using standard colorimetric assays. Agonist peptides
according to the invention provide at least about 10% of the activity of AAP in such
an assay, such as at least about 20% activity, for example at least about 30% activity,
such as at least about 40% activity, for example at least about 50% of the activity
of AAP, preferably, at least about 70% activity, and still more preferably, 100% or
greater activity of the activity of AAP.
Cancer
[0065] Preferred peptides according to the invention, alternatively, or additionally, decrease
GJIC inhibition mediated by tumor promoters such as DTT, in what is referred to herein
as a "standard tumor promoter assay." Preferably, the peptides show decreases in GJIC
inhibition, which are at least 50%, preferably 70%, and more preferably 100% or greater,
than decreases observed for AAP.
[0066] As discussed, it is an object of the invention to provide peptides that modulate
gap junction intercellular communication (GJIC). Thus, many peptides in accord with
the invention may include one or more of the following features: the ability to decrease
cellular uncoupling, to normalize dispersion of action potential duration, and to
normalize conduction velocity, the ability to control of the cellular quantity of
gap junctions normalizing (up-regulating or down-regulating as needed) the expression
of connexins; to normalize degradation of gap junctions (inhibit or enhance), to normalize
cellular trafficking of connexins to the plasma membrane (increase or decrease); to
facilitate assembly of connexins into functional gap junctions; to normalize opening
of existing gap junctions, e.g., inducing or enhancing opening when they have been
closed or gated by inhibitors (e.g., such as by mediating or enhancing hyperphosphorylation
of the cytoplasmic carboxy terminal domain of one or more connexins (e.g., such as
Cx43)) or closing these when they are aberrantly opened (e.g., as in Charcot-Marie-Tooth
disease); and the like.
[0067] Particular assays useful for identifying and optionally quantifying the activity
of preferred invention peptides are described below. The assays are non-limiting and
are merely serving the purpose of illustrating a variety of assays in which the present
peptides may be tested for their gap junction modulating abilities. It is to be understood
that the assays are not mutually exclusive, i.e. a peptide according to the invention
may show activity in one particular assay, but show a different, or no, activity in
another particular assay. This may be a reflection of the diversity of the individual
peptides and types of assays for testing said peptides.
A. Standard Oral Availability Assay
[0068] It is a preferred aspect of the invention to provide peptides with enhanced availability
in vivo. Absorption of peptides after oral administration is often limited because they are
degraded by either enzymes in the gastrointestinal (GI) tract or by enzymes in the
intracellular lumen of the enterocytes. Further, the physico-chemical properties of
peptides, especially their large hydrogen-bonding potential, makes it difficult for
these molecules to permeate the enterocytes by transcellular passive diffusion. However,
di- and tri-peptides, which have survived the gastrointestinal fluid enzymes can enter
the intracellular lumen by means of the peptide transporter hPepT1, which is a membrane
protein localized in the apical membrane of the enterocytes of the upper small intestine.
[0069] Preferred peptides according to the invention are therefore peptides that have affinity
for an hPepT1 transporter or an analog thereof. The three-dimensional conformation
and key binding sites of peptide compounds which bind the PepT1 transporter are described
by Bailey, P.D., et al., 2000,
supra, and desired peptides can be modeled
in silico to optimally fit within this binding site, as described above (see, e.g., Bailey,
P.D., et al., (2000),
supra;
Vinter, J.G., (1996) J. Comput. Aided Des. 10: 417).
[0070] The oral availability of a peptide comprising a structure as shown in Formula I,
Formula II, Table 1, or more generally identified using the Bailey assay, may be evaluated
for its ability to bind to a PepT1 transporter, preferably an hPepT1 transporter.
For example, a PepT1 cDNA, preferably, an hPepT1 cDNA (see, e.g.,
Covitz, K. M., et al., (1996) Pharm. Res. 13(11): 1631-34) may be expressed in a
Xenopus oocyte expression system and the uptake of labeled peptide into the oocyte can be
monitored to approximate Ki values as described in
Temple, C.S., et al. (1996) J. Physiol. (London) 494: 795;
Meredith, D., et al., (1998) J. Physiol. (London) 512: 629.
[0071] In one aspect of the invention, a standard
in vivo oral availability assay is performed to determine the oral availability of a peptide
which has been modeled to optimally conform to the Bailey substrate template, as discussed
above. In this assay, a peptide is orally administered to a mammal, such as a rat,
in anorally administrable form (e.g., as part of a food pellet or in water), while
at the same time, the same concentration of peptide is administered i.v. (e.g., through
a catheter inserted into the femoral vein and artery). Peptides may be administered
as bolus injections at concentrations ranging from 10
-5-10
-10 in volumes of 1 ml/kg for both oral and i.v. dosing. The animals are given 500 I.U.
of heparin i.v. 5 minutes before the first blood sample is taken. A control blood
sample, "before dose" or B.D. sample, is collected approximately 5 minutes before
administration of the peptides. A sample of the dosing solution (e.g., 100 µl of water
comprising 10
-5-10
-10 M of peptide) is retained for concentration determination. Blood samples are collected
at t=B.D. 5, 15, 30, 60, 90, 120, 180, and 240 minutes.
[0072] Blood is collected in labeled ice-chilled EDTA stabilized blood sample vials and
stored on ice until quickly centrifuged at 4°C for 5 minutes (10,000 x g). Plasma
(100 µl) is harvested, transferred to a labeled polypropylene microcentrifuge vial
(e.g., 0.5 ml eppendorf), frozen on ice and stored at -20°C until further analysis.
Approximately 40 µl of the filtrate is injected onto an HPLC column (XterraMS C18,
3 x 50 mm, 3.5 µm particles) and eluted using a linear gradient from 0 to 100% B in
4.0 minutes. The column is washed for 2.9 minutes in buffer B (0.1% formic acid in
acetonitrile or another suitable buffer) and equilibrated for 5 minutes in Buffer
A (0.1 % formic acid in water or another suitable buffer) prior to the next injection
of sample. Mass spectrometry is performed using methods routine in the art and as
described further below in Examples 1 and 2.
[0073] The concentrations of compounds in the plasma samples are calculated from an external
standard curve covering the range from 1.00 to 1000 nM. The plasma concentrations
versus time data are used for pharmacokinetic modeling in WinNonLin 3.5 (Pharsight,
Mountain view, CA) using non-compartmental analysis and AUC values are determined
as is known in the art. Preferably, orally available peptides according to the invention
are observed in significant levels In plasma within about 30 minutes or less. AUC
values observed in animals receiving i.v. administrations of peptide are used to evaluate
such effects as clearance and half-life which should be the same in the two systems.
B. Standard Plasma Stability Assays
[0074] The invention also provides peptides that have enhanced stability
In vitro or
In vivo. In one aspect the peptide comprises a peptide bond that is alkylated or otherwise
modified to stabilize the peptide against enzymatic degradation. In another aspect,
the peptide comprises one or more D-amino acids. In a further aspect, the peptide
has enhanced stability in a standard stability assay.
[0076] As disclosed in
WO02/077017, peptides can be incubated in plasma or serum and samples taken at regular intervals
for analysis by HPLC or LC/MS/MS, to quantitate the amount of undegraded peptide.
Appropriate conditions (column, solvent, gradient, and temperature) for such analyses
are estimated to ensure that the peptide peak and the plasma peaks do not have the
same retention time. This is done by subsequent Injections of a peptide, plasma, and
a co-injection with the peptide and the plasma, followed by optimization of LC method
parameters until a satisfactory separation is obtained. A control plasma sample without
the peptide, treated in the same manner, also can be taken and evaluated. The samples
may include, but are not limited to, a blank, the peptide at a suitable concentration
(e.g., 0,1 mg/mL), plasma without peptide, one or more samples for t = 0, and one
or more samples at each regular interval. Preferably, multiple samples are taken in
parallel. The sample concentrations (peak height in mAU or ion counts) can be plotted
vs, time and fitted to a function describing a mono exponential decay (e.g., using
a standard Excel package). Preferably, a peptide according to the invention has a
half-life of more than about 48 hours, such as more than 24 hours, for example more
than 12 hours, such as more than 6 hours, for example more than 3 hours, such as more
than 1 hour, for example more than 30 minutes as determined using this assay.
[0077] Plasma stability can be examined
in vivo using standard assays. For example, peptides may be administered to a mammal, such
as a rat, by bolus injections in volumes of about 1 ml/kg for both i.v. and p.o. dosing.
Preferably, peptides are tested in parallel with control samples such as buffer or
an antiarrythmic peptide with a known stability. Blood samples are collected at different
time periods (e.g., at B.D. 5, 15, 30, 60, 90, 120, 180, and 240 minutes, where B.D.
refers to before dose). Amounts of peptides in samples can be quantitated using methods
routine in the art, such as LC/MS/MS. For example, the concentrations of peptides
in plasma samples may be calculated from an external standard curve covering ranges
of peptide from 1.00 to 1000 nM. The plasma concentrations versus time data can be
used for pharmacokinetic modeling in WinNonLin 3.5 (Pharsight, Mountain view, CA)
using non-compartmental analysis and the resulting parameters of AUC, Fpo, Clb, t1/2,
Cmax and tmax can be determined as is known in the art.
C. Standard Cardiomyocyte Assays
[0078] In one aspect, a peptide according to the invention is administered to a cardiac
cell and gap junction function is evaluated. Optimal peptides for such procedures
can be identified in standard cardiomycte assays. In one aspect, cardiac cells are
isolated from a mammal, such as a guinea pig hearts by perfusion with collagenase
according to the Langendorf method. The cells are exposed to peptide and evaluated
for GJIC by patch clamp using methods known in the art. Intercellular conductance
(Gj) using the formula:
[0079] Where lp,pulse and lp,rest represent the current in the passive cell during the pulse
and before the pulse respectively, and Up and Ua represent the voltage of the passive
and active cell. The change in Gj value upon peptide administration is analyzed by
comparing the relative changes in Gj. For example, the relative Gj as a function of
time before, and during, stimulation with peptide (e.g., at about 10
-8 M) can be determined.
[0080] In one embodiment the peptide provides a Gj, which is substantially the same as the
Gj (by ± 10%) of an antiarrhythmic peptide such as AAP, AAP10, HP5, and functional
analogs thereof. In one aspect, the cell is an ischemic cell, and the peptide provides
a Gj, which is substantially the same as that of a non-ischemic cell (±20%, preferably,
± 10%).
[0081] In another embodiment the present peptides provides a GJ, which is different from
the Gj (by ± 40%) of an antiarrhytmic peptide such as AAP, AAP10, HP5, and functional
analogs thereof.
D. Standard Caiclum-induced Arrhythmia Assay
[0083] Peptides suitable for administration to cardiac cells can be identified in an
in vivo model of calcium-induced arrhythmias according to the model of
Lynch et al. (1981) J Cardiovasc.Pharmacol. 3; 49-60. Mice (25-30 g) are anaesthetized with a neurolept anaesthetic combination and an
i.v. cannula is inserted into the tail vein. A lead II ECG signal is recorded continuously
by positioning a stainless steel ECG electrodes on the right forelimb and on the left
hind limb. The ground electrode is placed on the right hind limb. The signal is amplified
(x 5.000-10.000) and filtered (0.1-150 Hz) via a Hugo Sachs Electronic model 689 ECG
module. The analog signal Is digitized via a 12 bit data acquisition board (Data Translation
model DT321) and sampled at 1000 Hz using the Notocord HEM 3.1 software for Windows
NT. After a 10-minute equilibration period, the test sample of peptide is injected
into the tail vein. Mice pre-treated with buffer are tested as a measure of the control
level In untreated animals. The injection volume is 100 µl in all experiments.
[0084] Infusion of CaCl
2 (30 mg/ml, 0.1 ml/min ≈ 100 mg/kg/min (calcium chloride-2-hydrate, Riedel-de Haën,
Germany)) is started 3 min after i.v. administration of drug or vehicle. The time
lag to onset of 2nd degree AV-block is determined as the time from the start of CaCl2
infusion until the first arrhythmic event occurs. An event of 2nd degree AV-block
Is defined as intermittent failure of the AV conduction characterised by a P-wave
without the concomitant QRS complex.
[0085] Responses are expressed relative to the time until 2nd degree AV-block occurred In
vehicle treated mice. The maximal effect of compounds (e.g., peptides, AAP, AAP10
or controls) is determined. Preferably, peptides according to the invention have antiarrhythmic
activity comparable to the compounds AAP, AAP10, HP5, or a functional analog thereof,
i.e., the peptides increase the time to an AV block in a mouse after Infusion of CaCl
2. Preferably, Preferably, the peptides provide at least about 40% of the activity
of AAP, for example at least about 50% of the activity of AAP, such as about 60% of
the activity of AAP, for example at least about 70% of the activity of AAP, such as
at least about 80% of the activity of AAP, for example at least about 90% of the activity
of AAP, for example at least about substantially the same activity of AAP, such as
about 110% of the activity of AAP, for example at least about 120% of the activity
of AAP, such as at least about 130% of the activity of AAP, for example at least about
140% of the activity of AAP, such as about 150% of the activity of AAP, for example
at least about 160% of the activity of AAP, such as at least about 170% of the activity
of AAP, for example at least about 180% of the activity of AAP, preferably at least
about 190% of the activity of AAP, more preferably at least about 200 or greater %
of the activity of AAP (i.e., the peptides show time lags of approximately the same
duration as induced by AAP).
E. Standard Osteoblast Activity Assay
[0086] Modulation of intercellular communication represents a mechanism by which osteotropic
factors regulate the activity of bone forming cells. Therefore, in one aspect, peptides
according to the invention are used to increase osteoblast activity by increasing
gap junctional communication between bone cells, thereby enhancing bone formation
in vivo.
[0087] The efficacy of a peptide according to the invention may be assayed in preliminarily
in human osteblast cells (hOB), for example by measuring calcium wave activity and/or
alkaline phosphatase activity.
[0088] In one aspect, cells are isolated from human bone marrow obtained by puncture of
the posterior iliac spine of healthy volunteers (aged 20-36): 10-15 ml marrow material
was collected in 15 ml PBS+Ca,Mg (Life Technologies, Cat. No. 14040) with 100 U/ml
Heparin (Sigma, Cat. No. H-3149). The mononuclear fraction of the marrow is isolated
on a Lymphoprep gradient (Nycomed Pharma, Cat.No. 1001967), by centrifugation at 2200
rpm for 30 min. After harvesting, the mononuclear fraction is washed once with culture
medium and centrifuged at 1800 rpm for 10 min. Subsequently cells are counted and
plated in culture medium at 5x10
6 cells/100 mm dish. hOB medium (all reagents obtained from Life Technologies): MEM
w/o Phenol Red w/ Glutamax (Cat.No. 041-93013) supplemented with 10% heat inactivated
fetal calf serum (Cat.No. 10106) and 0.1% Penicillin/Streptomycin (Cat.No. 15140).
Medium is changed the following day and the cells are cultured at 37°C in 5%CO
2 with medium change every 7 days. After 4-6 weeks of culture, the cells will reach
70% confluence. The medium is then supplemented with 100 nM Dexamethasone (Sigma,
Cat.No. D-4902) for 7 days. Cells are then plated for video imaging experiments: a
25 mm #1 glass coverslip is placed in a 35 mm dish (or each well of a 6-well multidish),
cells are plated at 2.5x10
5 cells/coverslip and cultured for 2-3 days before use.
[0089] ROS 17/2.8 cells are cultured in 100 mm dishes at 37°C with 5% CO
2 and medium changed every 2-3 days. ROS medium (all reagents obtained from Life Technologies):
MEM (Cat.No. 31095) is supplemented with 10% heat-inactivated calf serum (Cat.No.
16170), 1% NEAA (Cat.No. 11140), 1% Sodium Pyruvate (Cat.No. 11360), 1% L-Glutamine
(Cat.No. 25030) and 0.1% Penicillin/Streptomycin (Cat.No. 15140). For video imaging
experiments, cells are plated on coverslips at 2-3x10
5 cells/coverslip and cultured for 2-3 days before use.
[0090] The cells cultured on coverslips are loaded with 5 µM fura-2-AM (Molecular Probes,
Cat.No. F-1221), for 30 minutes at 37°C, and incubated in fresh medium for 20 minutes.
Coverslips are then affixed to a PDMI-2 culture chamber (Medical Systems Corp.), maintained
at 37°C with superfused CO
2, on a Zeiss Axiovert microscope. Intercellular calcium waves are induced by mechanical
stimulation of a single cell using a borosilicate glass micro pipette affixed to an
Eppendorf 5171 micromanipulator.
[0091] Imaging is performed using a MetaMorph imaging system (Universal Imaging). The excitation
light (340 and 380 nm) is provided by a monochromator (T.I.L.L. Photonics GmbH). Images
are acquired with an intensified CCD camera (Dage MTI) and digitized with a Matrox
MVP image processing board. The number of cells involved in a calcium wave in the
presence and absence of peptide can be used to provide a measure of increase in GJIC.
[0092] In one aspect, administration of a peptide increases the number of cells involved
in a wave at least about two-fold compared to cells which have been exposed to a control,
such as buffer. In another aspect, administration of a peptide decreases the number
of cells involved in a wave by at least about two-fold. Agonist peptides according
to the invention provide at least about 10% of the activity of AAP in such an assay,
such as at least about 20% activity, for example at least about 30% activity, such
as at least about 40% activity, for example at least about 50% of the activity of
AAP, preferably, at least about 70% activity, and still more preferably, 100% or greater
activity of the activity of AAP.
[0093] Cells also can be measured for the presence of alkaline phosphatase activity to provide
a general measure of osteoblast activity. In one aspect, cells are plated in 96-well
plates at a concentration of 8000 cells/well (hOB) or 3000 cells/well (ROS) in 200µl
normal culture medium. On day 4 (or day 3 for ROS cells), cells are washed with 200
µl MEM, 0.1% BSA (Sigma, Cat.No. A-9418). Samples comprising a suitable medium (e.g.,
200 µl MEM, 0.1% BSA) containing various concentrations of peptides, control, AAP
or AAP10 are added to the cells, and culture is continued for about 4 days (2 days
for ROS cells).
[0094] On about day 8 (preferably day 5 for ROS cells), cells are assayed for alkaline phosphatase
using an Alkaline Phosphatase (ALP) assay such as is known in the art. ALP assays
are generally colorimetric endpoint methods for measuring enzyme activity, and can
be performed using an Alkaline Phosphatase Kit (Sigma, Cat.No. 104-LL). Preferably,
cells are washed once with 200 µl PBS+Ca, Mg, 100µl Alkaline Buffer Solution is added
to each well and the cells are incubated at 37°C for 10 minutes. 100 µl Substrate
Solution is subsequently added to each well and the plate is incubated at 37°C for
30 min. 100 µl 2.0 N NaOH is added to each well to stop the reaction. Absorbance is
measured using a plate reader at 405 nm.
[0095] Agonist peptides according to the invention provide at least about a 5% increase
in alkaline phosphatase production relative to isotonic saline, preferably, at least
about 10% increase in alkaline phosphatase production relative to isotonic saline,
and still more preferably a 15% or greater increase in alkaline phosphatase production
relative to isotonic saline. The increase in production of alkaline phosphatase is
a measure for increased activity of osteoblasts and accordingly a measure for an increase
in bone formation.
F. Standard Tumor Promotor Assay
[0096] The compound 1,1-bis(
p-chlorophenyl)-2,2,2-trichlorethane, also known as the insecticide DDT, is an inhibitor
of gap junctional communication, and has tumor promoting abilities. It inhibits cell-to-cell
communication by reducing the gap junction number and size, and exposure to DDT is
associated with decreased cellular levels of phosphorylated (active) forms of the
gap junction protein Cx43. These actions are considered pivotal for the compound's
oncogenic properties (
X. Guan, et al. (1996) Carcinogenesis, 17 1791-1798;
R. J. Ruch, et al. (1994) Carcinogenesis, 15 301-306);
B. V. Madhukar, et al. (1996) Cancer Lett. 106 117-123). As a means of monitoring the therapeutic efficacy of the peptides, the effects
of the peptides on DDT-induced uncoupling in human osteoblast cells can be determined.
Thus, in one aspect, peptides according to the invention are used to inhibit or prevent
tumor-promoter induced decreases of GJIC (
W. K. Hong, et al. (1997) Science, 278 1073-1077).
[0097] In one exemplary assay, human osteoblast cells are isolated from human bone marrow
obtained by puncture of the posterior iliac spine of healthy volunteers (aged 20-36).
Approximately 10-15 ml of bone marrow material are collected in 15 ml PBS + Ca, Mg
(Life Technologies, Cat.No. 14040) with 100 U/ml Heparin (Sigma, Cat.No. H-3149).
The mononuclear fraction of the marrow is isolated on a Lymphoprep gradient (Nycomed
Pharma, Cat.No. 1001967), by centrifugation at 2200 rpm for 30 minutes.
[0098] After harvesting, the mononuclear fraction iswashed once with culture medium and
centrifuged at 1800 rpm for 10 minutes. Subsequently cells are counted and plated
in culture medium at 8x10
6 cells/100 mm dish. Medium is changed the following day and the cells are cultured
at 37°C in 5%CO
2 with medium changes every 7 days. After 3-4 weeks of culture, the cells typically
reach 70% confluence. The medium is then supplemented with 100 nM Dexamethasone (Sigma,
Cat.No. D-4902) for 7 days. Cells are then plated for video imaging experiments. Generally,
cells are plated at 2.5x10
5 cells/coverslip and are cultured for 2-3 days before imaging.
[0099] After culturing, cells are affixed to a PDMI-2 culture chamber (Medical Systems Corp.),
maintained at 37°C with superfused CO
2, on a Zeiss Axiovert microscope. Microinjections are performed using a micropipette
is loaded with a 10 mM Lucifer Yellow solution (Sigma, Cat.No. L-0259). A cell in
the monolayer is carefully injected with LY for 30 seconds; the micropipette is removed
from the cell and after 30 seconds the number of cells that show dye transfer is counted.
For a subset of cell cultures, DDT is added to the medium in a final concentration
of 13 µM, and is left on for 60 minutes. Images of cells are acquired with an intensified
CCD camera (Dage MTI) and digitized with a Matrox MVP image processing board, using
the MetaMorph imaging software (Universal Imaging).
[0100] Under control conditions (no DDT treatment), the dye generally spreads to a median
of 14.5 cells (n=12). DDT-exposed cells typically show decreased cellular coupling
with a median of 7 (n=13).
[0101] Peptides are added to the bathing solution in a final concentration of 10
-8 mol/l, and after 10 minutes, another microinjection is performed. Preferably, agonist
peptides according to the invention show an increase in cell-to-cell dye transfer.
More preferably, this increase is significantly different from control samples (without
peptides) as determined using routine statistical tests, such as the Wilcoxon non-parametric
statistical test (with p<0.05). Preferably, the peptides show decreases in GJIC inhibition
which are at least about 50%, preferably about 70%, and more preferably, about 100%
or greater, than decreases observed for AAP.
[0102] This assay can be used to identify candidate peptides with the highest therapeutic
efficacy in reversing the decreased intercellular coupling related to tumor promotion
and in one aspect, such peptides are administered to patients at risk for developing
or having cancer. A peptide may be used alone or in combination with other peptides
and/or in a combination therapy with other anti-cancer agents.
[0103] Still other assays may be performed to identify peptides which elicit substantially
the same physiological responses as the antiarrhythmic peptides AAP, AAP10, HP, and
their functional analogs (e.g., to identify agonists) or which inhibit or suppress
these physiological responses (e.g., to identify antagonists). Suitable assays include,
but are not limited to: assays to measure cAMP formation in cells (e.g., CHO cells);
cAMP efficacy assays (e.g., measuring inhibition of forskoline-stimulated cAMP formation
of APP-like compounds in CHO cells); phosphoinositol turnover in cardiomyocytes (Meier
et al.) (
E. Meier, et al. (1997) Drug Development Research, 40: 1-16); and responses to glucose and oxygen deprivation.
[0104] A number of standard assays are detailed above. Additional assays are described in
PCT/US02/05773, filed February 22, 2002, the entirety of which is incorporated herein by reference. These assays are exemplary
only and other suitable assays that may be developed and become standardized are encompassed
within the scope of the invention.
Pharmaceutical Compositions
[0105] The peptides may be formulated in a pharmaceutical composition comprising one or
more of any of the peptides described above, in combination with a pharmaceutically
acceptable carrier and/or diluent. Such compositions are preferably in a form adapted
for oral administration.
[0106] Formulations for oral administration may be prepared in a manner well-known to the
person skilled in the art, e.g., as generally described in "
Remington's Pharmaceutical Sciences", 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing
Company, Easton, PA, U.S.A., 1985 and more recent editions and in the monographs in the "Drugs and the Pharmaceutical
Sciences" series, Marcel Dekker. In one preferred aspect, the compositions are in
the form of tablets, capsules, granules, pellets, troches, lozenges, and the like.
Suitable enteric formulations are described, e.g., in
U.S. Patent No. 5,350,741.
[0107] The pharmaceutical carrier or diluent employed may be a conventional solid or liquid
carrier. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin,
talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl
ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil,
phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.
[0108] Similarly, the carrier or diluent may include any sustained release material known
In the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with
a wax.
[0109] If a solid carrier is used for oral administration, the preparation may be tabletted,
placed in a hard gelatin capsule in powder or pellet form, or it can be in the form
of a troche or lozenge. The amount of solid carrier will vary widely but will usually
be from about 25 mg to about 1 g.
[0110] If a liquid carrier is used, the preparation may be in the form of a syrup or liquid
suitable for oral ingestion.
[0111] It will be appreciated that the actual preferred amounts of active compounds used
in a given therapy will vary according to e.g. the specific compound being utilized,
the particular composition formulated, the mode of administration and characteristics
of the subject, e.g. the species, sex, weight, general health and age of the subject,
Optimal administration rates for a given protocol of administration can be readily
ascertained by those skilled in the art using conventional dosage determination tests
conducted with regard to the foregoing guidelines. Suitable dose ranges may include
from about 1 mg/kg to about 100mg/kg of body weight per day.
[0112] Therapeutic compounds of the invention are suitably administered in a protonated
and water-soluble form, e.g., as a pharmaceutically acceptable salt, typically an
acid addition salt such as an inorganic acid addition salt, e.g., a hydrochloride,
sulfate, or phosphate salt, or as an organic acid addition salt such as an acetate,
maleate, fumarate, tartrate, or citrate salt. Pharmaceutically acceptable salts of
therapeutic compounds of the invention also can include metal salts, particularly
alkali metal salts such as a sodium salt or potassium salt; alkaline earth metal salts
such as a magnesium or calcium salt; ammonium salts such an ammonium or tetramethyl
ammonium salt; or an amino acid addition salts such as a lysine, glycine, or phenylalanine
salt.
[0113] The compounds of the invention may also be administered topically to treat peripheral
vascular diseases and as such may be formulated as a cream or ointment.
[0114] The present peptides may also be formulated in compositions such as sterile solutions
or suspensions for non-oral administration. Peptides of the invention may be administered
parenterally, that is by intravenous, intramuscular, subcutaneous, intranasal, intrarectal,
intravaginal or intrapritoneal administration.
[0115] The present invention will now be further described by the following working examples,
which illustrate preferred embodiments of the invention.
Treatment Methods
[0116] In one aspect, the invention provides a method of administering to a patient having,
or at risk of developing, a condition associated with impaired GJIC, a therapeutically
effective amount of any of the peptides described above. Preferably, administration
is oral. Patients who may be treated using peptides according to the invention include,
but are not limited to, animals, preferably mammals, e.g., rodents (including mice,
rats, hamsters, and lagomorphs, such as rabbits), dogs, pigs, goats (generally any
domestic animal), and primates. In one preferred aspect, a patient is a human being.
[0117] In another aspect the invention concerns the use of a peptide according to the invention
for the manufacture of a medicament for the treatment of a pathological condition
involving impaired gap junctional communication comprising administering to a patient
a therapeutically effective amount of said peptide.
[0118] Examples of conditions which can be treated include, but are not limited to, cardiovascular
disease, inflammation of airway epithelium, disorders of alveolar tissue, bladder
incontinence, impaired hearing due to diseases of the cochlea, endothelial lesions,
diabetic retinopathy and diabetic neuropathy, ischemia of the central nervous system
and spinal cord, dental tissue disorders including periodontal disease, kidney diseases,
failures of bone marrow transplantation, wounds, erectile dysfunction, urinary bladder
incontinence, neuropathic pain, subchronic and chronic inflammation, cancer and failures
of bone marrow and stem cell transplantation, conditions which arise during transplantation
of cells and tissues or during medical procedures such as surgery; as well as conditions
caused by an excess of reactive oxygen species and/or free radicals and/or nitric
oxide.
Arrhythmia
[0119] In one preferred aspect, the invention provides a pharmacologically active antiarrhythmic
peptide, and the use thereof, for treatment of arrhythmias and thrombotic complications
arising during cardiovascular disorders, such as acute ischemic heart disease (e.g.,
stable angina pectoris, unstable angina pectoris, acute myocardial infarction), congestive
heart failure (e.g., systolic, diastolic, high-output, low-output, right or left sided
heart fallure), congenital heart diseases, cor pulmonale, cardiomyopathies, myocarditis,
hypertensive heart disease, during coronary revascularization, and the like.
[0120] In specific embodiments, an antiarrhythmic peptide according to the present invention
is used to treat and/or prevent bradyarrhythmias (e.g., due to disease in sinus node,
AV node, bundle of His, right or left bundle branch), and tachyarrhythmias associated
with reentry (e.g., atrial premature complexes, AV junctional complexes, ventricular
premature complexes, atrial fibrillation, atrial flutter, paroxymal supraventricular
tachycardia, sinus node reentrant tachycardia, AV nodal reentrant tachycardia, and
non-sustained ventricular tachycardia) either alone or in combination with other antiarrhythmic
compounds, such as class I agents (e.g., lidocaine), class II agents (e.g., metoprolol
or propranolol), class III agents (e.g., amiodarone or sotalol) or class IV agents
(e.g., verapamil).
[0121] Additionally, or alternatively, peptides according to the invention are used to treat
one or more of: a reentry arrhythmia; ventricular reentry (e.g., such as arises during
acute myocardial infarction, chronic myocardial infarction, stable angina pectoris
and unstable angina pectoris); infectious or autonomic cardiomyopathy; atrial fibrillation;repolarization
alternans; monomorphic ventricular tachycardia; T-wave alternans; bradyarrhythmias;
and generally, reduced contractility of cardiac tissue, thrombosis and the like.
Osteoporosis
[0122] In a further aspect, peptides according to the invention are used to prevent and/or
treat osteoporosis or other pathologies affecting bone formation, growth or maintenance.
Peptides which are able to normalize the attenuated GJIC between human osteoblast
during hypoxia are particularly suitable for the treatment of bone diseases with impaired
bone formation relative to bone resorption. Optimal peptides for use in such methods
can be selected in assays for increased alkaline phosphatase (ALP) activity in osteoblasts,
which provides a means to monitor cell viability and growth as a consequence of proper
maintenance of GJIC. In one aspect, human osteoblasts are stimulated with different
concentrations of peptides from 1 x 10
-13 to 1 x 10
-6, and compared to untreated controls. Under normal culture conditions, peptides preferably
increase ALP activity. Even more preferably, the peptides stimulate ALP activity during
hypoxic conditions at concentrations ranging from 10
-11 to 10
-8 mol/l. The assay can thus be used to optimize peptide compositions for the treatment
and/or prevention of bone diseases associated with poor vascularization, hypoxia and
ischemia in bone tissue.
[0123] In another aspect, peptides according to the invention are used for the prevention
and/or treatment of joint diseases that involves impaired cell-to-cell coupling. For
example, the peptides can be used for the prevention and/or treatment of joint diseases
that involve metabolic stress. These would include any form of arthritis associated
with decreased vascularization or healing of fractured cartilage tissue.
Cancer
[0124] In still another aspect, peptides according to the invention are used to treat cancer.
Carcinogenesis is characterized by the progressive impairment of growth control mechanisms
in which growth factors, oncogenes and tumor suppressor genes are involved. A general
theme in carcinogenesis and tumorigenesis is the down regulation of the GJIC. Permeability
of gap junctions in tumor cells using the dye-transfer assay is typically lower than
GJIC in surrounding tissue. Further, the gating of gap junctions is known to be effected
by tumor promoters, which decrease GJIC. Therefore, in one aspect, peptides according
to the invention are used as medicaments for the treatment of cancer, alone, or in
conjunction with traditional anti-cancer therapies.
Wounds
[0125] In a further aspect, peptides according to the invention are used to treat wounds
and, in particular, to accelerate wound healing. Wound healing involves the interactions
of many cell types, and intercellular communication mediated by gap junctions is considered
to play an important role in the coordination of cellular metabolism during the growth
and development of cells involved in tissue repair and regeneration (
K. M. Abdullah, et al. (1999) Endocrine, 10 35-41;
M. Saitoh, et al. (1997) Carcinogenesis, 18: 1319-1328;
J. A. Goliger, et al. (1995) Mol.Biol.Cell, 6 1491-1501). Peptides may be administered to the site of a wound by topical administration using
carriers well known in the art (e.g., ointments, creams, etc.) or may administered
systemically, e.g., for treating wounds of internal tissues, such as in the treatment
of chronic gastric ulcer lesions.
[0126] Additional functions in which endothelial gap-junctional intercellular communication
has been implicated are the migratory behavior of endothelial cells after injury,
angiogenesis, endothelial growth and senescence, and the coordination of vasomotor
responses (
G. J. Christ, et al. (2000) Braz. J Med Biol.Res., 33: 423-429). Therefore, in one aspect, a peptide according to the invention is used to enhance
conducted vascular responses and to improve blood supply during conditions with increased
metabolic demand (e.g., physical exercise, tachycardia), and during ischemia.
[0127] Gap junctions are also believed to provide the molecular link for co-ordinated long-range
signaling among individual members of the glial compartment. Likewise, astrocytes
are ideally suited for metabolic support of neurons since they are functionally polarized
with one extremity touching the vascular bed and the other pole approximates neuronal
parenchyma (
R. Dermietzel (1998) Brain Res. Brain Res. Rev., 26: 176-183). Therefore, in one preferred embodiment, peptides according to the invention are
administered to a patient in need to prevent ischemic damage in the brain by increasing
the metabolic support between glia cells and neurons. Such patients may include patients
with organic psychoses, which may present with signs such as depression, anxiety,
learning and memory deficit, phobias, and hallucinations or patients who have suffered
a traumatic brain injury. Preferably, such peptides are selected or formulated so
as to be available to the central nervous system (i.e., provided with or conjugated
with carriers which facilitate transport across the blood-brain barrier).
[0128] Peptides according to the invention may also be used to accelerate repair after nerve
injury or during grafting of immature cells (progenitor cells) into brain tissue,
e.g., such as in patients with neurotrauma, brain ischemia and chronic neurodegenerative
diseases, such as Parkinson's disease or Huntington's disease (
H. Aldskogius, et al. (1998) Prog. Neurobiol, 55: 1-26).
[0130] It should be obvious to those of skill in the art, that the peptides and pharmaceutical
compositions according to the invention can be used to treat any condition or pathology
associated with impaired (abnormal decreases or increases in) gap junctional communication.
Preferably, one or more of the peptides or pharmaceutical compositions comprising
the one or more peptides are administered to a patient in need thereof in a therapeutically
effective amount. As used herein, "a therapeutically effective amount" is one which
reduces symptoms of a given condition or pathology, and preferably which normalizes
physiological responses in a patient with the condition or pathology. Reduction of
symptoms or normalization of physiological responses can be determined using methods
routine in the art and may vary with a given condition or pathology. In one aspect,
a therapeutically effective amount of one or more peptides or pharmaceutical composition
comprising the one or more peptides is an amount which restores a measurable physiological
parameter to substantially the same value (preferably to within ± 30%, more preferably
to within ± 20%, and still more preferably, to within 10% of the value) of the parameter
in a patient without the DMF.
EXAMPLES
[0131] The invention will now be further illustrated with reference to the following examples.
It will be appreciated that what follows is by way of example only and that modifications
to detail may be made while still falling within the scope of the invention.
Example 1. Peptide Synthesis
[0132] A preferred general procedure is described below. However, more detailed descriptions
of solid phase peptide syntheses are found in
WO98/11125 hereby incorporated in its entirety.
a. General Peptide Synthesis
Apparatus and synthetic strategy
[0133] Peptides were synthesized batchwise in a polyethylene vessel equipped with a polypropylene
filter for filtration using 9-fluorenylmethyloxycarbonyl (Fmoc) as N-α-amino protecting
group and suitable common protection groups for side-chain functionalities.
Solvents
[0134] Solvent DMF (
N,N-dimethylformamide, Riedel de-Häen, Germany) was purified by passing through a column
packed with a strong cation exchange resin (Lewatit S 100 MB/H strong acid, Bayer
AG Leverkusen, Germany) and analyzed for free amines prior to use by addition of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine
(Dhbt-OH) giving rise to a yellow color (Dhbt-O
- anion) if free amines are present. Solvent DCM (dichloromethane, analytical grade,
Riedel de-Häen, Germany) was used directly without purification. Acetonitril ( HPLC-grade,
Lab-Scan, Dublin Ireland) was used directly without purification.
Amino Acids
[0135] Fmoc-protected amino acids were purchased from Advanced ChemTech (ACT) in suitabel
side-chain protected forms. Otherwise protected amino acids (Boc-Asp(OFm)-OH, Boc-D-Asp(OFm)-OH,
Fmoc-Lys(Aloc)-OH, Fmoc-D-Lys(Aloc)-OH, Boc-Lys(Fmoc)-OH Boc-D-Lys(Fmoc)-OH, Boc-Orn(Fmoc)-OH
from Bachem (Switzerland); Fmoc-Lys(ivDde)-OH, Fmoc-Sar-OH from NovaBiochem (Switzerland).
Benzoic acid and Benzyl Amine Derivatives
[0136] Benzoic acid and Benzyl amine derivatives were purchased from Aldrich and used without
further purification.
Coupling Reagents
[0137] Coupling reagent diisopropylcarbodiimide (DIC) was purchased from (Riedel de-Häen,
Germany), PyBop from Advanced ChemTech.
Linkers
[0138] (4-hydroxymethylphenoxy)acetic acid (HMPA), was purchased from Novabiochem, Switzerland;
and was coupled to the resin as a preformed 1-hydroxybenzotriazole (HOBt) ester generated
by means of DIC.
Solid Supports
[0139] Peptides synthesized according to the Fmoc-strategy on TentaGel S resins 0,22-0,31
mmol/g (TentaGel-S-NH
2; TentaGel S-Ram, Rapp polymere, Germany).
Catalysts and Other Reagents
[0140] Diisopropylethylamine (DIEA) was purchased from Aldrich, Germany, and ethylenediamine
from Fluka, piperidine and pyridine from Riedel-de Häen, Frankfurt, Germany. 4-(N,N-dimethylamino)pyridine
(DMAP) was purchased from Fluka, Switzerland and used as a catalyst in coupling reactions
involving symmetrical anhydrides. Ethandithiol was purchased from Riedel-de Häen,
Frankfurt, Germany. 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH), 1-hydroxybenzotriazole
(HOBt) (HOAt) were obtained from Fluka, Switzerland.
Coupling Procedures
[0141] The first amino acid can be coupled as a symmetrical anhydride in DMF generated from
the appropriate n-α-protected amino acid and the subsequent amino acids can be coupled
as
in situ generated HOBt or HOAt esters made from appropriate n-α-protected amino acids and
HOBt or HOAt by means of DIC in DMF. The acylations were checked by the ninhydrin
test performed at 80 oc in order to prevent Fmoc deprotection during the test (
B. D. Larsen, A. Holm, Int.J Pept.Protein Res. 1994, 43 1-9).
Deprotection of the N-α-amino Protecting Group (Fmoc)
[0142] Deprotection of the Fmoc group was performed by treatment with 20% piperidine in
DMF (1x5 and 1x10 min.), followed by wash with DMF (5 x 15 ml, 5 min. each) until
no yellow color could be detected after addition of Dhbt-OH to the drained DMF.
Deprotection of Allyl/Aloc
[0143] A solution of 3 eq. Pd(PPh
3)
4 dissolved in 15-20 ml CHCI
3, AcOH, NMM (37:2:1) was added to the peptid resin. The treatment was continued for
three hours at room temperature accompanied by bubbling a stream of N
2 through the mixture.
Coupling Of Hobt-Esters
[0144] 3 eq.
N-α-amino protected amino acid was dissolved in DMF together with 3 eq. HOBt and 3
eq. DIC and then added to the resin.
Preformed Symmetrical Anhydride
[0145] Six eq.
N-α-amino protected amino acid was dissolved in DCM and cooled to 0°C. DIC (3 eq.)
was added and the reaction continued for 10 minutes. The solvent was removed in vacuo
and the remanence dissolved in DMF. The solution was immediately added to the resin
followed by 0.1 eq. of DMAP.
Cleavage Of Peptide From Resin With Acid
[0146] Peptides were cleaved from the resins by treatment with 95% triflouroacetic acid
(TFA, Riedel-de Häen, Frankfurt, Germany)-water v/v or with 95% TFA and 5% ethandithiol
v/v at r.t. for 2 hours. The filtered resins were washed with 95% TFA-water and filtrates
and washings evaporated under reduced pressure. The residue was washed with ether
and freeze-dried from acetic acid-water. The crude freeze-dried product was analyzed
by high-performance liquid chromatography (HPLC) and identified by electrospray ionisation
mass spectrometry (ESMS).
Batchwise Peptide Synthesis on TentaGel Resin (PEG-PS)
[0147] TentaGel resin (1g, 0.22-0.31 mmol/g) was placed in a polyethylene vessel equipped
with a polypropylene filter for filtration. The resin was swelled in DMF (15ml), and
treated with 20% piperidine in DMF to secure the presence of non-protonated amino
groups on the resin. The resin was drained and washed with DMF until no yellow color
could be detected after addition of Dhbt-OH to the drained DMF. HMPA (3 eq.) was coupled
as a preformed HOBt-ester as described above and the coupling was continued for 24
h. The resin was drained and washed with DMF (5 x 5 ml, 5 min each) and the acylation
checked by the ninhydrin test. The first amino acid was coupled as a preformed symmetrical
anhydride as described above. The following amino acids according to the sequence
were coupled as preformed Fmoc-protected HOBt esters (3 eq.) as described above. The
couplings were continued for 2 h, unless otherwise specified. The resin was drained
and washed with DMF (5 x 15 ml, 5 min each) in order to remove excess reagent. All
acylations were checked by the ninhydrin test performed at 80 °C. After completed
synthesis the peptide-resin was washed with DMF (3x15 ml, 5 min each), DCM (3x15 ml,
1 min each) and finally diethyl ether (3x15 ml, 1 min each) and dried
in vacuo.
[0148] Preparative HPLC conditions.
[0149] Preparative chromatography was carried out using a VISION Workstation (PerSeptive
Biosystem) equipped with AFC2000 automatic fraction collector/autosampler. VISION-3
software was used for instrument control and data acquisition.
Column
[0150] Kromasil (EKA Chemicals) KR100-10-C8 100A, C-8, 10 □; CER 2230, 250 x 50,8 mm or
a VYDAC 218TP101550, 300A, C-18, 10-15 □, 250 x 50 mm. The buffer system used included
A: 0,1% TFA in MQV; B: 0,085% TFA, 10% MQV, 90% MeCN. Flow rates were 35-40 ml/min
and the column temperature was 25°C. UV detection was performed at 215 nm and 280
nm. Suitable gradients were optimized for individual peptides.
Analytical HPLC Conditions
[0151] Gradient HPLC analysis was done using a Hewlett Packard HP 1100 HPLC system consisting
of a HP 1100 Quaternary Pump, a HP 1100 Autosampler a HP 1100 Column Thermostat and
HP 1100 Multiple Wavelength Detector. Hewlett Packard Chemstation for LC software
(rev. A.06.01) was used for instrument control and data acquisition.
[0152] For analytical HPLC, different columns were used as appropriate, such as VYDAC 238TP5415,
C-18, 5µm, 300A, or a Jupiter, Phenomenex 00F-4053-E0; 5 □m C-18, 300A 150 x 4,6 mm
and others. The buffer system included A: 0,1% TFA in MQV; B: 0,085% TFA, 10% MQV,
90% MeCN. Flow rates were 1 ml/min. The preferred column temperature was 40°C. UV
detection was performed at 215 nm. As above, suitable gradients were optimized for
the individual peptides.
Mass Spectroscopy
[0153] The peptides were dissolved in super gradient methanol (Labscan, Dublin, Ireland),
Milli-Q water (Millipore, Bedford, MA) and formic acid (Merck, Damstadt, Germany)
(50:50:0.1 v/v/v) to give concentrations between 1 and 10 µg/m). The peptide solutions
(20 µl) were analysed in positive polarity mode by ESI-TOF-MS using a LCT mass spectrometer
(Micromass, Manchester, UK) accuracy of +/- 0.1 m/z.
General synthetic procedure
[0154] In all syntheses, dry TentaGel-S-NH
2 (0.23 mmol/g, 1g) was placed in a polyethylene vessel equipped with a polypropylene
filter for filtration and treated as described under "batchwise peptide synthesis
on TentaGel resin". Lysine and analogs thereof (e.g. Ornithin, 2,4-Diaminobutanoic
acid, 1,3-diaminopropanoic acid etc.) when situated C-terminally were coupled as the
N-α, Fmoc protected derivatives with either ivDde or Aloc protection of the side chain
functionality (e.g. Fmoc-Lys(Aloc)-OH). Lysine and analogs thereof when situated N-terminally
were coupled as the N-α Boc protected derivatives with Fmoc protection of the side
chain functionality (e.g. Boc-Lys(Fmoc)-OH). Aspartic acid, Glutamic acid and analogs
thereof when situated C-terminally were coupled as N-α Fmoc protected derivatives
with Allylic protection of the side chain functionality (e.g. Fmoc-Asp(Oallyl)-OH).
Aspartic acid, Glutamic acid and analogs thereof when situated N-terminally were coupled
as N-α Boc protected derivatives with Fm protection of the side chain functionality
(e.g. Boc-Asp(OFm)-OH). Other amino acids than the above mentioned when situated C-terminally
were coupled as N-α Fmoc protected derivatives with suitable protection of the side
chain functionalities or when situated N-terminally as N-α Boc protected derivatives
with suitable protection of the side chain functionalities.
[0155] In all cases the dipeptide was assembled followed by deprotection of the side chain
protecting group of the Lysine, Aspartic- or Glutamic acid or analogs thereof.
[0156] In case of Lysine or analogs thereof, the hydrophobic group functionalised as a carboxylic
acid was coupled as an
in situ generated HOBt ester by means of DIC in THF.
[0157] In case of Aspartic- and Glutamic acid or analogs thereof, the hydrophobic group
functionalised as an amine was coupled to the pre generated HOBt ester of the side
chain carboxylic acid by means of DIC in DMF catalyzed by triethylamine.
[0158] All couplings were continued for at least 2 hours. The acylations were checked by
the ninhydrin test performed at 80 °C as earlier described. After completed synthesis
the peptide-resin was washed with DMF (3x 15 ml, 1 min each), DCM (3x 15 ml, 1 min
each), diethyl ether (3x 15 ml, 1 min each) and dried
in vacuo. The peptide was then cleaved from the resin as described above and freeze-dried.
[0159] After purification using preparative HPLC as described above, the peptide product
was collected and the identity of the peptide was confirmed by ES-MS .
[0160] The above procedure was used for the synthesis of all peptides exemplified further
below and the peptides shown in Table 1 of the specification. Exemplary synthesis
schemes are shown in Figures 1A and 1B.
b. synthesis of Individual Peptides
Synthesis of H-Gly-Lys (4-nitrobenzoyl)-OH (Compound 1)
[0161] Lysine was coupled as Fmoc-Lys(ivDde)-OH and the N-terminal Glycine as the Boc derivative.
The ivDde protecting group was removed as described above. The Lysine and Glycine
were first assembled on the solid support. The ivDdde group was then removed and subsequently
4-nitrobenzoic acid was coupled as an
in situ generated HOBt ester by means of DIC in THF. After purification using preparative
HPLC as described above, 35 mg peptide product was collected with a purity greater
than 99 %. The identity of the peptide was confirmed by ES-MS (found MH
+ 352.28, calculated MH
+ 352.21).
Synthesis of H-Gly-Lys(4-cyanobenzoyl)-OH (Compound 3)
[0162] Lysine was coupled as Fmoc-Lys(ivDde)-OH and the N-terminal Glycine as the Boc derivative.
The ivDde protecting group was removed as described above. The Lysine and the Glycine
were first assembled on the solid support. The ivDdde group was then removed and subsequently
4-cyanobenzoic acid was coupled as an
in situ generated HOBt ester by means of DIC in DMF. After purification using preparative
HPLC as described above, 6 mg peptide product was collected with a purity greater
than 90 %. The identity of the peptide was confirmed by ES-MS (found MH
+ 332.25, calculated MH
+ 332.21).
Synthesis of H-Gly-Lys(4-methoxybenzoyl)-OH (Compound 4)
[0163] Lysine was coupled as Fmoc-Lys(ivDde)-OH and the N-terminal Glycine as the Boc derivative.
The ivDde protecting group was removed as described above. The Lysine and the Glycine
were first assembled on the solid support. The ivDdde group was then removed and subsequently
4-methoxybenzoic acid was coupled as an
in situ generated HOBt ester by means of DIC in DMF. After purification using preparative
HPLC as described above, 7 mg peptide product was collected with a purity greater
than 95 %. The identity of the peptide was confirmed by ES-MS (found MH
+ 337.28, calculated MH
+ 337.21).
Synthesis of H-Lys(4-nitrobenzoyl)-Gly-OH (Compound 7)
[0164] Glycine was coupled as Fmoc-Glycine and the N-terminal Lysine was coupled as Boc-Lys(Fmoc)-OH.
The Glycine and the Lysine were first assembled on the solid support. The
[0165] Fmoc in the side chain of Lysine was then removed and subsequently 4-nitrobenzoic
acid was coupled as an
in situ generated HOBt ester by means of DIC in THF. After purification using preparative
HPLC as described above, 64 mg peptide product was collected with a purity greater
than 98 %. The identity of the peptide was confirmed by ES-MS (found MH
+ 352.19, calculated MH
+ 352.24).
Synthesis of H-D-Lys(4-methoxybenzoyl)-Gly-OH (Compound 21)
[0166] Glycine was coupled as Fmoc-Glycine and the N-terminal Lysine was coupled as Boc-D-Lys(Fmoc)-OH.
The Glycine and the Lysine were first assembled on the solid support. The Fmoc in
the side chain of Lysine was then removed and subsequently 4-methoxybenzoic acid was
coupled as an
in situ generated HOBt ester by means of DIC in DMF. After purification using preparative
HPLC as described above, 31 mg peptide product was collected with a purity greater
than 97 %. The identity of the peptide was confirmed by ES-MS (found MH
+ 337.24, calculated MH
+ 337.21).
Synthesis of H-D-Lys(4-nitrobenzoyl)-Gly-OH (Compound 22)
[0167] Glycine was coupled as Fmoc-Glycine and the N-terminal Lysine was coupled as Boc-D-Lys(Fmoc)-OH.
The Glycine and the Lysine were first assembled on the solid support. The Fmoc in
the side chain of Lysine was then removed and subsequently 4-nitrobenzoic acid was
coupled as an
in situ generated HOBt ester by means of DIC in THF. After purification using preparative
HPLC as described above, 65 mg peptide product was collected with a purity greater
than 98 %. The identity of the peptide was confirmed by ES-MS (found MH+ 352.27, calculated
MH+ 352.24).
Synthesis of H-D-Lys(benzoyl)-Gly-OH (Compound 23)
[0168] Glycine was coupled as Fmoc-Glycine and the N-terminal Lysine was coupled as Boc-D-Lys(Fmoc)-OH.
The Glycine and the Lysine were first assembled on the solid support. The Fmoc in
the side chain of Lysine was then removed and subsequently Benzoic acid was coupled
as an
in situ generated HOBt ester by means of DIC in DMF. After purification using preparative
HPLC as described above, 31 mg peptide product was collected with a purity greater
than 96 %. The identity of the peptide was confirmed by ES-MS (found MH+ 307.22, calculated
MH+ 307.24).
Synthesis of H-D-Lys(4-t-Butylbenzoyl)-Gly-OH (Compound 54)
[0169] Glycine was coupled as Fmoc-Glycine and the N-terminal Lysine was coupled as Boc-D-Lys(Fmoc)-OH.
The Glycine and the Lysine were first assembled on the solid support. The Fmoc in
the side chain of Lysine was then removed and subsequently 4-t-Butylbenzoic acid was
coupled as an
in situ generated HOBt ester by means of DIC in DMF. After purification using preparative
HPLC as described above, 34 mg peptide product was collected with a purity greater
than 97 %. The identity of the peptide was confirmed by ES-MS (found MH+ 363.27, calculated
MH+ 363.22).
Synthesis of H-Asn(NH(4-methoxybenzyl))- D-Alanine-OH (Compound 80)
[0170] Alanine was coupled as Fmoc-D-Alanine and the N-terminal Asparagine was coupled as
Boc-Asp(OFm)-OH. The Alanine and the Aspartic acid were first assembled on the solid
support. The Fm in the side chain of Aspartic acid was then removed and subsequently
4-methoxybenzylamine was coupled catalyzed by triethylamine to the pre generated HOBt
ester of the side chain carboxylic acid by means of DIC in DMF. After purification
using preparative HPLC as described above, 17 mg peptide product was collected with
a purity greater than 98 %. The identity of the peptide was confirmed by ES-MS (found
MH+ 323.17, calculated MH+ 323.15).
Synthesis of H-D-Asn(NH(4-methoxybenzyl))-Alanine-OH (Compound 95)
[0171] Alanine was coupled as Fmoc-Alanine and the N-terminal Asparagine was coupled as
Boc-D-Asp(OFm)-OH. The Alanine and the Aspartic acid were first assembled on the solid
support. The Fm in the side chain of Aspartic acid was then removed and subsequently
4-methoxybenzylamine was coupled catalyzed by triethylamine to the pre generated HOBt
ester of the side chain carboxylic acid by means of DIC in DMF. After purification
using preparative HPLC as described above, 25 mg peptide product was collected with
a purity greater than 98 %. The identity of the peptide was confirmed by ES-MS (found
MH+ 323.12, calculated MH+ 323.15).
Synthesis of H-D-Asn(NH(4-nitrobenzyl))-Alanine-OH (Compound 96)
[0172] Alanine was coupled as Fmoc-Alanine and the N-terminal Asparagine was coupled as
Boc-D-Asp(OFm)-OH. The Alanine and the Aspartic acid were first assembled on the solid
support. The Fm in the side chain of Aspartic acid was then removed and subsequently
4-nitrobenzylamine was coupled catalyzed by triethylamine to the pre generated HOBt
ester of the side chain carboxylic acid by means of DIC in DMF. After purification
using preparative HPLC as described above, 102 mg peptide product was collected with
a purity greater than 99 %. The identity of the peptide was confirmed by ES-MS (found
MH+ 338.14, calculated MH+ 338.12).
Example 2 Bioavailability
MATERIALS AND METHODS
Pharmacokinetic screening and food interaction studies
Chemicals & Materials
[0173] The water used for these experiments was of highest quality obtained from a reversed
osmosis primary system in combination with a Milli-Q water secondary treatment system
(Millipore, Bedford, MA, USA). Methanol was super gradient quality obtained from Labscan
Ltd. (Dublin, Ireland). Formic acid p.a. (98-100%) was obtained from Merck (Darmstadt,
Germany). Heptafluorobuturicacid, HPLC grade wAs obtained from Pierce (Rockford, III,
USA). EDTA stabilised plasma from rat (Sprague-Dawley) was obtained from Harlan Sera
Lab Ltd. (Loughborough, UK). Blood samples were collected in potassium EDTA coated
microtainers from BD Vacutainer Systems (Plymouth, UK). Sample preparation by ultra
filtration was performed using Microcon centrifugal filter devices with a molecular
weight cut off of 3000 obtained from Millipore (Bedford, MA, USA).
Instrumentation
[0174] The LC/MS/MS analysis was performed on a Waters Alliance 2790 HPLC instrument in
combination with a Quattro Ultima mass spectrometer from Micromass (Manchester, UK).
Both the LC and MS were controlled by MassLynx 3.5 software. The LC separations prior
to MS/MS detection were performed on an XTerra MS C
18 (2.0 x 50 mm), 3.5 µm particles, (Waters, Milford, MA, USA).
Animals
[0175] Twelve male Sprague-Dawley rats (approx 350 g) were obtained from M&B (Denmark) and
catheters were inserted into the femoral vein and artery during Hypnorm
®-Dormicum
® anaesthesia. After surgery, the rats were allowed to recover for five days before
drug administration was initiated.
Drugs and Dose Levels
[0176] The dipeptides (Compound 22, Compound 21, Compound 23, Compound 96, Compound 95 and
Compound 54) were obtained as the TFA salts and dissolved in PBS (2.6 mM KCI, 137
mM NaCI, 1.5 mM KH
2PO
4 and 8.2 mM Na
2HPO
4 adjusted to pH=7.4) to give concentrations of 500 µM. The dosing volumes were 1 mL/kg
corresponding to doses of 500 nmol/kg, respectively. After each experiment the dosing
solutions were diluted in 0.5% formic acid in water and analysed by LC/MS/MS for their
content of drug substance. The concentrations of drug substance in the dosing solutions
were calculated from the responses of standards in the range from 1 to 1000 nM.
Study Design
[0177] The drug substances were administered to two animals as i.v. and p.o. bolus in a
cross-over study design. The p.o. dose was administered to rats fasted for a 17 hours
period prior to drug administration. The animals were allowed to rest for 48 hours
in between drug administrations. After the first experiment the rats received a blood
transfusion with heparin treated blood.
[0178] Prior to drug administration (5 min) the rats received 500 IU heparin as an i.v.
bolus and a control blood sample was collected. After drug administration blood samples
of approx. 250 µL were collected from the femoral artery at the following time points;
before dose (B.D.), 5, 15, 30, 60, 120, 180 and 240 min after i.v. administration
and at B.D., 10, 30, 50, 80, 120, 180, 240, and 300 min after p.o. administration.
The blood samples were stored on ice until centrifugation for 5 min at 10.000 x g
(4°C) and the plasma (100 µL) were transferred to 1.5 mL polypropylene tubes and stored
at -20°C until sample preparation and LC/MS/MS analysis.
Sample Preparation and LC/MS/MS Analysis
[0179] The plasma samples were thawed on ice, and 100 µL plasma was mixed with 100 µL 1
% (v/v) formic acid and transferred to microcon YM-3 filter units. The samples were
then centrifuged for 1 hr at 8.300 x g at room temperature. The filtrates were collected
in 300 µL autosampler vials and stored at 4°C until injection (40 µL) onto the LC
column. The chromatography was performed at 50°C using a linear gradient from 100
% buffer A (0.1% formic acid in water) to 100% buffer B (0.1% formic acid in acetonitrile)
in 4 min, followed by 3 min wash using buffer B and finally a reequilibration period
for 7 min using buffer A. The detection of the drug substances were performed by MS/MS
using argon as collision gas (1.3 x 10
-3 mbar). The specific MS/MS settings for each of the tested compounds are given in
Table 2.
Table 2: MS/MS settings used for the detection of compounds x-y in the bioavailability study.
Compound |
Cone voltage |
Collision Energy |
Parent ion |
Daughter ion(s) |
22 |
40 V |
24 eV |
353.15 m/z |
150.00 m/z |
21 |
60 V |
28 eV |
338.15 m/z |
135.00 m/z |
23 |
30 V |
18 eV |
308.10 m/z |
187.9 m/z |
104.9 m/z |
96 |
45 V |
15 eV |
339.15 m/z |
220.00 m/z |
169.80 m/z |
123.90 m/z |
95 |
60 V |
20 eV |
324.10 m/z |
120.90 m/z |
54 |
60 V |
26 eV |
364.20 m/z |
160.90 m/z |
Data Analysis
[0180] The plasma concentrations of the drug substance were calculated from the area related
to an external calibration curve obtained from spiked plasma samples treated as described
above. The calibration curve was obtained by linear regression of the log(peak area)
vs. log(concentration). The calibration curve covered the concentration range from
0.1 to 100 nM.
[0181] The plasma concentrations versus time data from each animal was used for pharmacokinetic
modelling in WinNonLin 3.5 (Pharsight, Mountain View, CA, USA) using non-compartmental
analysis and the value of the area under the plasma concentrartion curve (AUC) were
reported. The oral bioavailability F
po was calculated as the ratio in % between the dose (D) normalised AUC's after iv and
po administration: AUC
po*D
iv/AUC
iv*D
po.
RESULTS
[0182] The oral bioavailability of 6 compounds was tested and all compounds were detected
after oral administration. The calculated bioavailabilities are summarised in Table
3. The bioavailability of Compounds 22, 23, 96, and 95 were moderate (within 10-31%)
and the bioavailability of Compounds 54 and 21 were low (within 2 - 8%).
Table 3: The mean bioavailability ± standard error on mean (SEM) of the 6 tested compounds.
Compound No. |
F (%) |
|
Mean |
SEM |
22 |
31 |
5.3 |
21 |
2.6 |
0.78 |
23 |
10 |
5.7 |
96 |
15 |
3.0 |
95 |
29 |
10 |
54 |
7.2 |
0.40 |
CONCLUSION
[0183] The peptides of the invention showed oral availability having a range of availability
in Sprague-Dawley rats between 2 an 31%.
Example 3 Osteoblast Activity (ALP assay)
MATERIALS AND METHODS
[0184] Human primary osteoblasts were obtained from bone marrow biopsies of the posterior
iliac spine from voluntary donors. Cells were used at 6 weeks of culturing. Cells
were cultured in 24-well plates in an 8 days process and stimulated with the test
compounds in concentrations from 1x10
-13 to 1x10
-6 for the last 4 days of the experiment. Cell lysate was collected for determination
of ALP activity. hOB cells from 10-15 donors were used for the experiments. For ALP
measurements a commercially available kit (Alkaline Phosphatase Kit, Sigma-Aldrich
Denmark A/S) was used. All determinations were performed according to the manufacturer's
recommendations.
Analyses
[0185] ALP was determined on cell lysate collected at termination of the experiment. Measurements
were compared with and to vehicle treated control cultures.
Data analysis
[0186] Data are presented as means and standard deviations or medians and percentiles as
appropriate. Two-way ANOVA is performed using Fisher's LSD test for post-hoc comparisons,
using a 0.05 level of significance.
RESULTS
[0187]
Table 4
Compound |
N |
Mean |
SD |
0.05 int |
22 |
4 |
1,12 |
0,05 |
0,05 |
1 |
4 |
1,08 |
0,07 |
0,06 |
23 |
5 |
1,08 |
0,07 |
0,06 |
95 |
5 |
1,14 |
0,09 |
0,08 |
[0188] The activity of Compounds 1, 22, 23, and 95 are illustrated in Figure 2 and summarized
in the above Table 4.
CONCLUSION
[0189] The peptides of the invention increased the activity of human osteoblasts in primary
culture as measured by an increase in production of alkaline phosphatase.
Example 4 Standard Calcium-Induced Arrhythmia Assay
[0190] The anti-arrhythmic effect of the dipeptides was determined in a model of calcium-induced
arrhythmias according to the model of Lynch
et al. [6]. Male NMRI mice (25-30 g; Bomholdtgaard, Ll. Skendsved, Denmark) were anaesthetised
with a neurolept anaesthetic combination (Hypnorm
® (fentanyl citrate 0.315 mg/ml and fuanisone 10 mg/ml) + midazolam (5 mg/ml)). Commercial
solutions of Hypnorm
® and midazolam were diluted 1:1 in distilled water, and one part diluted Hypnorm
® was mixed with one part diluted midazolam. Anaesthesia was induced by s.c. administration
of this solution in a dose of 50 - 75 µl/10 gram mouse.
[0191] An i.v. cannula was inserted into the tail vein. The lead II ECG signal was recorded
continuously by positioning of stainless steel ECG electrodes on the right forelimb
and on the left hind limb. The ground electrode was placed on the right hind limb.
The signal was amplified (x 5.000-10.000) and filtered (0.1-150 Hz) via a Hugo Sachs
Electronic model 689 ECG module. The analogue signal was digitised via a 12-bit data
acquisition board (Data Translation model DT321) and sampled at 1000 Hz using the
Notocord HEM 3.1 software for Windows NT. After a 10-min equilibration period, the
test sample of drug was injected into the tail vein and three minutes later i.v. infusion
of CaCl
2 (30 mg/ml, 0.1 ml/min ≈ 100 mg/kg/min), (calciumchlorid-2-hydrat, Riedel-de Haën,
Germany) was started.
[0192] Mice pre-treated with vehicle (phosphate buffered saline with 0.1% bovine albumin)
were tested on all days as a measure for the control level in untreated animals. Injection
volume was 100 µl in all experiments. The time lag to onset of arrhythmias (t
arr) was determined as the time from the start of CaCl
2 infusion until the first event of 2
nd degree AV block (defined as intermittent failure of the AV conduction characterised
by a P-wave without the concomitant QRS complex).
RESULTS
[0193] The % response of the tested compounds are given in Table 5. The given response is
estimated according to (t
arr (testcompound) - t
arr (vehicle)) x 100/ t
arr (vehicle).
Table 5
Compound |
% response |
SEM |
1 |
52.0 |
8.0 |
3 |
37.0 |
9.0 |
4 |
69.0 |
11.0 |
7 |
40 |
7 |
21 |
55.0 |
4.0 |
22 |
52.0 |
12.0 |
23 |
-8.0 |
7.0 |
54 |
61.3 |
12.3 |
80 |
33 |
6 |
95 |
0.3 |
9.4 |
96 |
54 |
6 |
AAP |
18.9 |
6.4 |
CONCLUSION
[0194] The peptides of the invention showed both an antiarrhytmic effect and a non-antiarrhytmic
effect.
DEFINITIONS
[0195] Unless specified otherwise, the following definitions are provided for specific terms
which are used in the following written description.
[0196] Throughout the description and claims the three-letter code for natural amino acids
is used as well as generally accepted three letter codes for other α-amino acids,
such as Sarcosin (Sar). Where the L or D form has not been specified, it is to be
understood that the amino acid in question can be either the L or D form.
[0197] The term "halogen" refers to F, Cl, Br, and I, where F and I are preferred.
[0198] The term "alkyl" refers to univalent groups derived from alkanes by removal of a
hydrogen atom from any carbon atom: C
nH
2n+1-. The groups derived by removal of a hydrogen atom from a terminal carbon atom of
unbranched alkanes form a subclass of normal alkyl (n-alkyl) groups: H[CH
2]
n-. The groups RCH
2-, R
2CH- (R not equal to H), and R
3C- (R not equal to H) are primary, secondary and tertiary alkyl groups respectively.
C(1-22)alkyl refers to any alkyl group having from 1 to 22 carbon atoms and includes
C(1-6)alkyl, such as methyl, ethyl, propyl, iso-propyl, butyl, pentyl and hexyl and
all possible isomers thereof.
[0199] By the phrase "lower alkyl" is meant a linear or branched alkyl having less than
about 6 carbon atoms, preferably methyl, ethyl, propyl, or butyl.
[0200] The term "alkenyl" refers to a straight or branched or cyclic hydrocarbon group containing
one or more carbon-carbon double bonds. C(2-22)alkenyl refers to any alkenyl group
having from 1 to 22 carbon atoms and includes C(2-6)alkenyl, vinyl, allyl, 1-butenyl,
etc.
[0201] The term "aralkyl" refers to aryl C(1-22)alkyl, and the term "aryl" throughout this
specification means phenyl or naphthyl.
[0202] By the phrase "hydrophobic group" is meant an optionally substituted aromatic carbon
ring, preferably a 6 or 12 -membered aromatic carbon ring. By the phrase "optionally
substituted" is meant substitution of the 6 or 12-membered aromatic carbon ring with
at least one of a lower alkyl, alkoxy, hydroxyl, carboxy, amine, thiol, hydrazide,
amide, halide, hydroxyl, ether, amine, nitrile, imine, nitro, sulfide, sulfoxide,
sulfone, thiol, aldehyde, keto, carboxy, ester, an amide group; including seleno and
thio derivatives thereof. Also included in the definition of "optionally substituted"
are are sulfide, sulfoxide, sulfone, and thiol derivates with or without a seleno
group. In embodiments in which the aromatic carbon ring is substituted such substitutions
will typically number less than about 10 substitutions, more preferably about 1 to
to 5 of same with about 1 or 2 substitutions being preferred for many invention applications.
Preferred alkoxy groups include methoxy, ethoxy, and propoxy. Illustrative hydrophobic
groups include unsubstituted benzyl, phenyl, and napthyl.
[0203] By the phrase "hydrogen bond group" is meant a donor or acceptor of a (non-covalent)
hydrogen bond. In embodiments in which the hydrogen bond group is the donor, it will
typically include at least one electronegative atom bound to a hydrogen atom. Examples
of such electronegative atoms include, but are not limited to, nitrogen, oxygen, halide
(eg., Cl, F, Br, etc), and sulfur. Illustrative hydrogen bond donors include hydroxyl,
amine, thiol, hydrazide and amide. In embodiments in which the first hydrogen bond
group is an acceptor, it will preferably include at least one electronegative atom
such as those mentioned above in which the atom includes a non-bonded electron pair.
Such pairs are often referred to as "lone pairs". Examples of preferred hydrogen bond
acceptors include, but are not limited to, halide, hydroxyl, ether, amine, nitrile,
imine, nitro, aldehyde, keto, carboxy, ester, amide group; and seleno derivatives
thereof. Also envisioned are suitable sulfide, sulfoxide, sulfone, and thiol hydrogen
bond acceptors including seleno derivatives thereof.
[0204] The terms "intercellular communication modulator", "gap junction facilitator", "compound
that facilitates gap junction communication" and "gap junction opener", etc., all
refer to a compound that facilitates, or maintains, or normalizes (e.g. either by
inhibiting of enhancing), GJIC, irrespective of the particular mechanism behind this
action. More specifically, the term "gap junction opener" may refer to a substance
which normalizes (i.e., increases) the exchange of molecules that are able to pass
through gap junctions between extracellular and intracellular spaces and/or which
can normalize increase GJIC.
[0205] The term "agonist" refers to a peptide that can interact with a tissue, cell or cell
fraction which is the target of for example, but not limited to, an AAP, AAP10, HP5
peptide, or functional analog thereof, to cause substantially, or at least substantially
the same physiological responses in the tissue, cell or cell fractionas the AAP, AAP10,
HP5 peptide, or functional analog thereof. In one aspect, the physiological response
is one or more of: contraction, relaxation, secretion, and enzyme activation. Preferably,
the peptide binds to the tissue, cell or cell fraction. In one aspect, the peptide
binds to a receptor on the tissue, cell, or cell fraction, which binds to AAP, AAP10,
HP5, or a functional analog thereof. However, the present "agonist" may interact with
a tissue, cell or cell fraction, which is the target of any given peptide, causing
the same, or at least substantially the same physiological response.
[0206] An "antiarrhythmic peptide agonist" as used herein is a peptide, which comprises
an antiarrhythmic activity, which is substantially the same, or greater than, the
antiarrhythmic activity of an AAP, AAP10, HP5 peptide or functional analog thereof.
"Greater than" refers to an antiarrhythmic activity, which is observed at lower concentrations
of peptide or in shorter periods of time compared to the antiarrhythmic activity of
an AAP, AAP10, HP5 peptide or functional analog thereof.
[0207] The term "antagonist" refers to a peptide which inhibits or antagonizes one or more
physiological responses observed in a tissue, cell or cell fraction after contacting
the tissue, cell, or cell fraction with any given peptide, such as AAP, AAP10, HP5
peptide, or a functional analog thereof. In one aspect, the physiological response
is one or more of: contraction, relaxation, secretion, and enzyme activation. Preferably,
the peptide binds to the tissue, cell or cell fraction. In one aspect, the peptide
binds to a receptor on the tissue, cell, or cell fraction which binds to AAP, AAP10,
HP5, or a functional analog thereof and/or which inhibits binding of one or more of
AAP, AAP10, HP5, a functional analog thereof to the receptor.
[0208] As used herein, "normalize" refers to a change in a physiological response such that
the response becomes insignificantly different from one observed in a normal patient.
Thus, normalization may involve an increase or decrease in the response depending
on the pathology involved.
[0209] EC50 of less than about 10
-6M, and preferably, less than about, 10
-8 M.
[0210] As used herein "oral availability" refers to the rate and extent of absorption of
an orally administered drug into the blood stream.
1. A peptide for use in therapy represented by the general formula I:
wherein:
a is 1 and b is 0; or
b is 1 and a is 0;
d is 0-8; and
z is 1-7; and
x is 1, y and q are 1, and p is 0; or
p is 1, x and q are 0, and y is 1;
and further wherein,
if R1 is H then d is 0-8; or
if R1 is not H then d is 0;
wherein R1 is the side chain of an amino acid selected from the group consisting of alanine,
arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, and valine;
wherein R2 is selected from the group consisting of NH2, NHR, NR2, NR3+H, OH, SH, RO, RS, RSO, RSO2, COR, CSR, COOH, COOR, CONH2, CONHR, CONR2, OCOR, and SCOR, wherein R = alkyl, alkenyl, aryl, aralkyl, or cycloalkyl;
wherein R3 is H or CH3; and
wherein Rx is a hydrophobic group;
or a pharmaceutically acceptable salt thereof.
2. The peptide according to claim 1, wherein Rx comprises an aromatic carbon ring.
3. The peptide according to claim 2, wherein the aromatic ring comprises a 6- or 12 membered
ring or a substituted form thereof.
4. The peptide according to claim 3, wherein the ring is substituted with at least one
of: a lower alkyl, alkoxy, hydroxyl, carboxy, amine, thiol, hydrazide, amide, halide,
hydroxyl, ether, amine, nitrile, imine, nitro, sulfide, sulfoxide, sulfone, thiol,
aldehyde, keto, carboxy, ester, an amide group; a seleno group, a thio group and derivatives
thereof.
5. The peptide according to claim 3, wherein the ring comprises between 1 and 5 substitutions.
6. The peptide according to claim 3, wherein the ring comprises 1 or 2 substitutions.
7. The peptide according to claim 2, wherein the aromatic carbon ring is selected from
the group consisting of: a benzyl, phenyl, and naphthyl group.
8. The peptide according to claim 1 or claim 2, wherein the hydrophobic group is 6-membered
aromatic carbon ring comprising a substituent at the 4-position.
9. A peptide for use in therapy according to claim 1 represented by the general formula
II:
wherein:
a is 1 and b is 0; or
b is 1 and a is 0;
d is 0-8; and
z is 1-7; and
x is 1, y and q are 1, and p is 0; or
p is 1, x and q are 0, and y is 1;
and further wherein,
if R1 is H then d is 0-8; or if R1 is not H then d is 0;
wherein R1 is the side chain of an amino acid selected from the group consisting of alanine,
arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, and valine;
wherein R2 is selected from the group consisting of NH2, NHR, NR2, NR3+H, OH, SH, RO, RS, RSO, RSO2, COR. CSR, GOOH, COOR, CONH2, CONHR, CONR2, OCOR, and SCOR, wherein R = alkyl, alkenyl, aryl, aralkyl, or cycloalkyl;
wherein R3 is H or CH3;
wherein R4 and R5 are independently selected from the group consisting of H, alkyl, alkenyl, aryl,
aralkyl, halogen, CN, NO2, alkoxy, aryloxy, aralkyloxy, thioalkoxy, thioaryloxy, thioaralkyloxy, +S(CH3)2, SO3H, SO2R, NH2, NHR, NR2, +NR3, OH, SH, COOH, COOR, CONH2, CONHR, CONR2, CH2OH, NCO, NCOR, NHOH, NHNH2, NHNRH, CH2OCOR, CH2OCSR, COR, CSR, CSOR, CF3, and CCl3, and wherein R is alkyl, alkenyl, aryl, aralkyl, or cycloalkyl;
or a pharmaceutically acceptable salt thereof.
10. A peptide represented by the general formula I:
wherein:
a is 1 and b is 0; or
b is 1 and a is 0; and
d is 0-8; and
z is 1-7;
x is 1, y and q are 1, and p is 0; or
p is 1, x and q are 0, and y is 1;
and further wherein,
if R1 is H then d is 0-8; or
if R1 is not H then d is 0;
wherein R1 is the side chain of an amino acid selected from the group consisting of alanine,
arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, and valine;
wherein R2 is selected from the group consisting of NH2, NHR, NR2, NR3+H, OH, SH, RO, RS, RSO, RSO2, COR, CSR, COOH, COOR, CONH2, CONHR, CONR2, OCOR, and SCOR, wherein R = alkyl, alkenyl, aryl, aralkyl, or cycloalkyl;
wherein R3 = H or CH3; and
wherein Rx is a 6-membered aromatic carbon ring comprising a substituent at the 4-position;
or a pharmaceutically acceptable salt thereof.
11. The peptide according to claim 10, wherein the aromatic carbon ring is substituted
with at least one of: a lower alkyl, alkoxy, hydroxyl, carboxy, amine, thiol, hydrazide,
amide, halide, hydroxyl, ether, amine, nitrile, imine, nitro, sulfide, sulfoxide,
sulfone, thiol, aldehyde, keto, carboxy, ester, an amide group; a seleno group, a
thio group and derivatives thereof.
12. The peptide according to any one of the preceding claims, wherein the peptide comprises
a free N-terminal, a free C-terminal, or both a free N- and C-terminal.
13. The peptide of any one of the preceding claims, wherein the pharmaceutically acceptable
salt is an acid addition salt, a metal salt, an ammonium salt or an amino acid addition
salt.
14. The peptide of claim 13, wherein the pharmaceutically acceptable salt is a hydrochloride
salt, a sulfate salt, a phosphate salt, an acetate salt, a maleate salt, a fumarate
salt, a tartrate salt, a citrate shalt, a sodium salt, a potassium salt, a magnesium
salt, a calcium salt, an ammonium or tetramethyl ammonium salt,
a lysine salt, a glycine salt or a phenylalanine salt.
15. The peptide according to any one of the preceding claims, wherein the peptide further
comprises a hydrogen bond group and the distance between the mass center of the hydrogen
bond group and the hydrophobic group comprises from about 4 Angstroms to about 12
Angstrøms.
16. The peptide according to any one of the preceding claims, wherein the peptide further
comprises a hydrogen bond group and the distance between the mass center of the hydrogen
bond group and the hydrophobic group comprises from about 5 Angstrøms to about 10
Angstroms.
17. The peptide according to claim 10, wherein the substituent has a radius of from 3
to 11 Angstrøms.
18. The peptide according to claim 17, wherein the substituent is selected from the group
consisting of a methyl, ethyl, t-butyl, c-hexyl, phenyl, n-butyl, n-hexyl, n-octyl,
ethoxy, t-butoxy, phenoxy, butoxy, benzyloxy, n-hexyloxy, and n-octyloxy group.
19. The peptide according to any one of the preceding claims, wherein the peptide functions
as an antiarrhythmic drug.
20. The peptide according to any one of the preceding claims, wherein the peptide is an
orally available peptide.
21. The peptide according to any one of the preceding claims, wherein the peptide binds
to an hPepT1 transporter or a biologically active fragment thereof.
22. The peptide according to any one of the preceding claims, wherein the peptide has
a half-life in an in vitro plasma stability assay of more than 30 minutes.
23. The peptide according to any one of the preceding claims, wherein the peptide has
a half-life in an in vitro plasma stability assay of more than about 48 hours.
24. The peptide according to any one of the preceding claims, wherein the peptide comprises
a peptide bond that is modified to stabilize the peptide against enzymatic degradation.
25. The peptide according to any one of the preceding claims, wherein the peptide binds
to a tissue, cell, or cell fraction that is a site of action for an antiarrhythmic
peptide.
26. The peptide according to claim 23, wherein the antiarrhythmic peptide is selected
from the group consisting of AAP, AAP10, HP5, or a functional analog thereof.
27. The peptide according to claim 23, wherein the peptide is a modulator of the function
of the tissue, cell, or cell fraction.
28. The peptide according to claim 25, wherein the peptide antagonizes the function of
the antiarrhythmic peptide.
29. The peptide according to claim 25, wherein the peptide agonizes the function of the
antiarrhythmic.
30. The peptide according to claim 23, wherein the peptide is a modulator of a receptor
of the antiarrhythmic peptide.
31. The peptide according to any one of the preceding claims, wherein the peptide is selected
from the group consisting of the peptides shown in Table 1.
32. The peptide according to any one of the preceding claims, wherein the peptide is:
H-Gly-Lys(4-nitrobenzoyl)-OH (Compound 1);
H-Gly-Lys(4-methoxybenzoyl)-OH (Compound 4);
H-D-Lys(4-methoxybenzoyl)-Gly-OH (Compound 21);
H-D-Lys(4-nitrobenzoyl)Gly-OH (Compound 22);
H-D-Lys(4-t-butylbenzoyl)-Gly-OH (Compound 54);
H-D-Asn(NH(4-nitrobenzyl)Ala-OH (Compound 96);
H-D-Lys(benzoyl)Gly-OH (Compound 23) or
H-D-Asn(NH(4-methoxybenzyl)Ala-OH (Compound 95).
33. Use of a peptide according to any one of claims 1 to 32 in the manufacture of a medicament
for the treatment of a pathological condition selected from a cardiovascular disease,
inflammation of airway epithelium, a disorder of alveolar tissue, bladder incontinence,
impaired hearing, an endothelial lesion, diabetic retinopathy, diabetic neuropathy,
ischemia of the central nervous system, ischemia of the spinal cord, a dental tissue
disorder, kidney disease, failure of bone marrow transplantation, wound, erectile
dysfunction, urinary bladder incontinence, neuropathic pain, subchronic and chronic
inflammation, cancer, transplantation failure, osteoporosis or a pathology affecting
bone formation, growth or maintenance
34. The use according to claim 33, wherein the cardiovascular disease is selected from
arrhythmia, acute ischemic heart disease, congestive heart failure, congenital heart
diseases, cor pulmonale, cardiomyopathy, myocarditis or hypertensive heart disease.
35. The use according to claim 33 or claim 34, wherein the medicament is for oral administration.
36. The use according to any one of claims 33 to 35, wherein the medicament is for administration
to a human patient.
1. Peptid zur therapeutischen Verwendung, dargestellt durch die allgemeine Formel I:
worin:
a = 1 ist und b = 0 ist; oder
b = 1 ist und a = 0 ist;
d = 0 bis 8 ist; und
z = 1 bis 7 ist; und
x = 1 ist, y und q = 1 sind und p = 0 ist; oder
p = 1 ist, x und q = 0 sind und y = 1 ist;
und worin weiters,
wenn R1 = H ist, d = 0 bis 8 ist; oder,
wenn R1 nicht H ist, d = 0 ist;
worin R1 die Seitenkette einer aus der aus Alanin, Arginin, Asparagin, Asparaginsäure, Cystein,
Glutaminsäure, Glutamin, Glycin, Histidin, Isoleucin, Leucin, Lysin, Methionin, Phenylalanin,
Prolin, Serin, Threonin, Tryptophan, Tyrosin und Valin bestehenden Gruppe ausgewählten
Aminosäure ist;
worin R2 aus der aus NH2, NHR, NR2, NR3+H, OH, SH, RO, RS, RSO, RSO2 COR, CSR, COOH, COOR, CONH2, CONHR, CONR2, OCOR und SCOR bestehenden Gruppe ausgewählt ist und worin R = Alkyl, Alkenyl, Aryl,
Aralkyl oder Cycloalkyl ist;
worin R3 = H oder CH3 ist; und
worin Rx eine hydrophobe Gruppe ist;
oder ein pharmazeutisch annehmbares Salz davon.
2. Peptid nach Anspruch 1, worin Rx einen aromatischen Kohlenstoffring umfasst.
3. Peptid nach Anspruch 2, worin der aromatische Ring einen 6- oder 12-gliedrigen Ring
oder eine substituierte Form davon umfasst.
4. Peptid nach Anspruch 3, worin der Ring mit zumindest einem von einem Niederalkyl,
Alkoxy, Hydroxyl, Carboxy, Amin, Thiol, Hydrazid, Amid, Halogenid, Hydroxyl, Ether,
Amin, Nitril, Imin, Nitro, Sulfid, Sulfoxid, Sulfon, Thiol, Aldehyd, Keto, Carboxy,
Ester, einer Amidgruppe, einer Selenogruppe, einer Thiogruppe und Derivaten davon
substituiert ist.
5. Peptid nach Anspruch 3, worin der Ring zwischen 1 und 5 Substitutionen umfasst.
6. Peptid nach Anspruch 3, worin der Ring 1 oder 2 Substitutionen umfasst.
7. Peptid nach Anspruch 2, worin der aromatische Kohlenstoffring aus der aus einer Benzyl-,
einer Phenyl- und einer Naphthylgruppe bestehenden Gruppe ausgewählt ist.
8. Peptid nach Anspruch 1 oder Anspruch 2, worin die hydrophobe Gruppe ein 6-gliedriger
aromatischer Kohlenstoffring ist, der einen Substituenten an Position 4 umfasst.
9. Peptid zur therapeutischen Verwendung nach Anspruch 1, dargestellt durch die allgemeine
Formel II:
worin:
a = 1 ist und b = 0 ist; oder
b = 1 ist und a = 0 ist;
d = 0 bis 8 ist; und
z = 1 bis 7 ist; und
x = 1 ist, y und q = 1 sind und p = 0 ist; oder
p = 1 ist, x und q = 0 sind und y = 1 ist;
und worin weiters,
wenn R1 = H ist, d = 0 bis 8 ist; oder,
wenn R1 nicht H ist, d = 0 ist;
worin R1 die Seitenkette einer aus der aus Alanin, Arginin, Asparagin, Asparaginsäure, Cystein,
Glutaminsäure, Glutamin, Glycin, Histidin, Isoleucin, Leucin, Lysin, Methionin, Phenylalanin,
Prolin, Serin, Threonin, Tryptophan, Tyrosin und Valin bestehenden Gruppe ausgewählten
Aminosäure ist;
worin R2 aus der aus NH2, NHR, NR2, NR3+H, OH, SH, RO, RS, RSO, RSO2, COR, CSR, COOH, COOR, CONH2, CONHR, CONR2, OCOR und SCOR bestehenden Gruppe ausgewählt ist und worin R = Alkyl, Alkenyl, Aryl,
Aralkyl oder Cycloalkyl ist;
worin R3 = H oder CH3 ist; und
worin R4 und R5 unabhängig voneinander aus der aus H, Alkyl, Alkenyl, Aryl, Aralkyl, Halogen, CN,
NO2, Alkoxy, Aryloxy, Aralkyloxy, Thioalkoxy, Thioaryloxy, Thioaralkyloxy, +S(CH3)2, SO3H, SO2R, NH2, NHR, NR2, +NR3, OH, SH, COOH, COOR, CONH2, CONHR, CONR2, CH2OH, NCO, NCOR, NHOH, NHNH2, NHNRH, CH2OCOR, CH2OCSR, COR, CSR, CSOR, CF3 und CCl3 bestehenden Gruppe ausgewählt sind und worin R Alkyl, Alkenyl, Aryl, Aralkyl oder
Cycloalkyl ist; oder ein pharmazeutisch annehmbares Salz davon.
10. Peptid, dargestellt durch die allgemeine Formel I:
worin:
a = 1 ist und b = 0 ist; oder
b = 1 ist und a = 0 ist; und
d = 0 bis 8 ist; und
z = 1 bis 7 ist;
x = 1 ist, y und q = 1 sind und p = 0 ist; oder
p = 1 ist, x und q = 0 sind und y = 1 ist;
und worin weiters,
wenn R1 = H ist, d = 0 bis 8 ist; oder,
wenn R1 nicht H ist, d = 0 ist;
worin R1 die Seitenkette einer aus der aus Alanin, Arginin, Asparagin, Asparaginsäure, Cystein,
Glutaminsäure, Glutamin, Glycin, Histidin, Isoleucin, Leucin, Lysin, Methionin, Phenylalanin,
Prolin, Serin, Threonin, Tryptophan, Tyrosin und Valin bestehenden Gruppe ausgewählten
Aminosäure ist;
worin R2 aus der aus NH2, NHR, NR2, NR3+H, OH, SH, RO, RS, RSO, RSO2, COR, CSR, COOH, COOR, CONH2, CONHR, CONR2, OCOR und SCOR bestehenden Gruppe ausgewählt ist und worin R = Alkyl, Alkenyl, Aryl,
Aralkyl oder Cycloalkyl ist;
worin R3 = H oder CH3 ist; und
worin Rx ein 6-gliedriger aromatischer Kohlenstoffring ist, der einen Substituenten an Position
4 umfasst;
oder ein pharmazeutisch annehmbares Salz davon.
11. Peptid nach Anspruch 10, worin der aromatische Kohlenstoffring mit zumindest einem
von einem Niederalkyl, Alkoxy, Hydroxyl, Carboxy, Amin, Thiol, Hydrazid, Amid, Halogenid,
Hydroxyl, Ether, Amin, Nitril, Imin, Nitro, Sulfid, Sulfoxid, Sulfon, Thiol, Aldehyd,
Keto, Carboxy, Ester, einer Amidgruppe, einer Selenogruppe, einer Thiogruppe und Derivaten
davon substituiert ist.
12. Peptid nach einem der vorangegangenen Ansprüche, worin das Peptid einen freien N-Terminus,
einen freien C-Terminus oder sowohl einen freien N- als auch einen freien C-Terminus
umfasst.
13. Peptid nach einem der vorangegangenen Ansprüche, woran das pharmazeutisch annehmbare
Salz ein Säureadditionssalz, ein Metallsalz, ein Ammoniumsalz oder ein Aminosäureadditionssalz
ist.
14. Peptid nach Anspruch 13, worin das pharmazeutisch annehmbare Salz ein Hydrochloridsalz,
ein Sulfatsalz, ein Phosphatsalz, ein Acetatsalz, ein Maleatsalz, ein Fumaratsalz,
ein Tartratsalz, ein Citratsalz, ein Natriumsalz, ein Kaliumsalz, ein Magnesiumsalz,
ein Calciumsalz, ein Ammonium- oder Tetramethylammoniumsalz, ein Lysinsalz, ein Glycinsalz
oder ein Phenylalaninsalz ist.
15. Peptid nach einem der vorangegangenen Ansprüche, worin das Peptid weiters eine Wasserstoffbindungsgruppe
umfasst und die Entfernung zwischen dem Massenmittelpunkt der Wasserstoffbindungsgruppe
und der hydrophoben Gruppe etwa 4 Angström bis etwa 12 Angström umfasst.
16. Peptid nach einem der vorangegangenen Ansprüche, worin das Peptid weiters eine Wasserstoffbindungsgruppe
umfasst und die Entfernung zwischen dem Massenmittelpunkt der Wasserstoffbindungsgruppe
und der hydrophoben Gruppe etwa 5 Angström bis etwa 10 Angström umfasst.
17. Peptid nach Anspruch 10, worin der Substituent einen Radius von 3 bis 11 Angström
aufweist.
18. Peptid nach Anspruch 17, worin der Substituent aus der aus einer Methyl-, Ethyl-,
t-Butyl-, c-Hexyl-, Phenyl-, n-Butyl-, n-Hexyl-, n-Octyl-, Ethoxy-, t-Butoxy-, Phenoxy-,
Butoxy-, Benzyloxy-, n-Hexyloxy- und n-Octyloxy-Gruppe bestehenden Gruppe ausgewählt
ist.
19. Peptid nach einem der vorangegangenen Ansprüche, worin das Peptid als antiarrhythmisches
Arzneimittel fungiert.
20. Peptid nach einem der vorangegangenen Ansprüche, worin das Peptid ein oral verfügbares
Peptid ist.
21. Peptid nach einem der vorangegangenen Ansprüche, worin das Peptid an einen hPepT1-Transporter
oder ein biologisch aktives Fragment davon bindet.
22. Peptid nach einem der vorangegangenen Ansprüche, worin das Peptid in einem In-vitro-Plasmastabilitätstest
eine Halbwertszeit von mehr als 30 min aufweist.
23. Peptid nach einem der vorangegangenen Ansprüche, worin das Peptid in einem In-vitro-Plasmastabilitätstest
eine Halbwertszeit von mehr als 48 h aufweist.
24. Peptid nach einem der vorangegangenen Ansprüche, worin das Peptid eine Peptidbindung
umfasst, die so modifiziert ist, dass sie das Peptid gegen enzymatischen Abbau stabilisiert.
25. Peptid nach einem der vorangegangenen Ansprüche, worin das Peptid an ein Gewebe, eine
Zelle oder eine Zellfraktion bindet, das bzw. die eine Wirkstelle für ein antiarrythmisches
Peptid ist.
26. Peptid nach Anspruch 23, worin das antiarrhythmische Peptid aus der aus AAP, AAP10,
HP5 und einem funktionellen Analog davon bestehenden Gruppe ausgewählt ist.
27. Peptid nach Anspruch 25, worin das Peptid ein Modulator der Funktion des Gewebes,
der Zelle oder der Zellfraktion ist.
28. Peptid nach Anspruch 25, worin das Peptid der Funktion des antiarrhythmischen Peptids
entgegenwirkt.
29. Peptid nach Anspruch 25, worin das Peptid die Funktion des antiarrhythmischen Peptids
unterstützt.
30. Peptid nach Anspruch 23, worin das Peptid ein Modulator eines Rezeptors des antiarrhythmischen
Peptids ist.
31. Peptid nach einem der vorangegangenen Ansprüche, worin das Peptid aus der aus den
in Tabelle 1 gezeigten Peptiden bestehenden Gruppe ausgewählt ist.
32. Peptid nach einem der vorangegangenen Ansprüche, worin das Peptid:
H-Gly-Lys(4-nitrobenzoyl)-OH (Verbindung 1);
H-Gly-Lys(4-methoxybenzoyl)-OH (Verbindung 4);
H-D-Lys(4-methoxybenzoyl)Gly-OH (Verbindung 21);
H-D-Lys(4-nitrobenzoyl)Gly-OH (Verbindung 22);
H-D-Lys(4-t-butylbenzoyl)Gly-OH (Verbindung 54);
H-D-Asn(NH(4-nitrobenzoyl))Ala-OH (Verbindung 96);
H-D-Lys(benzoyl)Gly-OH (Verbindung 23) oder
H-D-Asn(NH(4-methoxybenzyl))Ala-OH (Verbindung 95) ist.
33. Verwendung eines Peptids nach einem der Ansprüche 1 bis 32 bei der Herstellung eines
Medikaments zur Behandlung eines aus einer Herz-Gefäß-Erkrankung, einer Entzündung
des Luftröhrenepithels, einer Erkrankung des Alveolargewebes, Blaseninkontinenz, Gehörschaden,
einer Endothelläsion, diabetischer Retinopathie, diabetischer Neuropathie, Ischämie
des Zentralnervensystems, Ischämie des Rückenmarks, einer Zahngewebeerkrankung, Nierenerkrankung,
gescheiterter Knochenmarktransplantation, einer Wunde, erektiler Dysfunktion, Harnblaseninkontinenz,
neuropathischem Schmerz, subchronischer und chronischer Entzündung, Krebs, gescheiterter
Transplantation, Osteoporose oder einer Pathologie, die die Bildung, das Wachstum
oder die Erhaltung des Knochens betrifft, ausgewählten pathologischen Leidens.
34. Verwendung nach Anspruch 33, worin die Herz-Gefäß-Erkrankung aus Arrythmie, akuter
ischämischer Herzerkrankung, kongestiver Herzinsuffizienz, kongenitalen Herzerkrankungen,
Cor pulmonale, Myokarditis oder hypertonischer Herzerkrankung ausgewählt ist.
35. Verwendung nach Anspruch 33 oder Anspruch 34, worin das Medikament zur oralen Verabreichung
dient.
36. Verwendung nach einem der Ansprüche 33 bis 35, worin das Medikament zur Verabreichung
an einen menschlichen Patienten dient.
1. Peptide pour utilisation en thérapie représenté par la formule générale I:
où:
a est 1 et b est 0; ou
b est 1 et a est 0;
d est 0-8; et
z est 1-7; et
x est 1, y et q sont 1, et p est 0; ou
p est 1, x et q sont 0, et y est 1;
et en outre où,
si R1 est H, alors d est 0-8; ou
si R1 n'est pas H, alors d est 0;
où R1 est la chaîne latérale d'un acide aminé sélectionné dans le groupe consistant en
alanine, arginine, asparagine, acide aspartique,
cystéine, acide glutamique, glutamine, glycine, histidine, isoleucine, leucine, lysine,
méthionine, phénylalanine, proline, sérine, thréonine, tryptophane, tyrosine et valine;
où R2 est sélectionné dans le groupe consistant en
NH2, NHR, NR2, NR3+H, OH, SN, RO, RS, RSO, RSO2, COR, CSR, COOH, COOR, CONH2, CONHR, CONR2, OCOR et SCOR, où R = alkyle, alkényle, aryle, aralkyle ou cycloalkyle;
où R3 est H ou CH3 et
où Rx est un groupe hyrophobe;
ou un sel pharmaceutiquement acceptable de celui-ci.
2. Peptide selon la revendication 1, où Rx comprend un cycle de carbone aromatique.
3. Peptide selon la revendication 2, où le cycle aromatique comprend un cycle à 6 ou
12 membres ou une forme substituée de celui-ci.
4. Peptide selon la revendication 3, où le cycle est substitué par au moins un de: alkyle
inférieur, alkoxy, hydroxyle, carboxy, amine, thiole, hydrazide, amide, halogénure,
hydroxyle, éther, amine, nitrile, imine, nitro, sulfure, sulfoxyde, sulfone, thiol,
aldéhyde, kéto, carboxy, ester, un groupe amide; un groupe séléno; un groupe thio
et leurs dérivés.
5. Peptide selon la revendication 3, où le cycle comprend entre 1 et 5 substitutions.
6. Peptide selon la revendication 3, où le cycle comprend 1 ou 2 substitutions.
7. Peptide selon la revendication 2, où le cycle de carbone aromatique est sélectionné
dans le groupe consistant en: un groupe benzyle, phényle et naphtyle.
8. Peptide selon la revendication 1 ou la revendication 2, où le groupe hydrophobe est
un cycle de carbone aromatique à 6 membres comprenant un substituant à la position
4.
9. Peptide pour utilisation en thérapie selon la revendication 1, représenté par la formule
générale II:
où:
a est 1 et b est 0; ou
b est 1 et a est 0;
d est 0-8; et
z est 1-7; et
x est 1, y et q sont 1, et p est 0; ou
p est 1, x et q sont 0, et y est 1;
et en outre où:
si R
1 est H, alors d est 0-8; ou
si R
1 n'est pas H, alors d est 0;
où R
1 est la chaîne latérale d'un acide aminé sélectionné dans le groupe consistant en
alanine, arginine, asparagine, acide aspartique, cystéine, acide glutamique, glutamine,
glycine, histidine, isoleucine, leucine, lysine, méthionine, phénylalanine, proline,
serine, threonine, tryptophan, tyrosine et valine;
où R
2 est sélectionné dans le groupe consistant en NH
2,
NHR, NR
2, NR
3+H, OH, SH, RO, RS, RSO, RSO
2, CO
R, CSR, COOH, COOR, CONH
2, CONHR, CONR
2, OCOR et SCOR, où R = alkyle, alkényle, aryle, aralkyle ou cycloalkyle:
où R3 is H or CH3;
où R4 et R5 sont indépendamment sélectionnés dans le groupe consistant en H, alkyle, alkényle,
aryle, aralkyle, halogène, CN, NO2, alkoxy, aryloxy, aralkyloxy, thioalkoxy, thioaryloxy, thioaralkyloxy, +S(CH3)2, SO3H, SO2R, NH2, NHR, NR2, +NR3, OH, SH, COOH, COOR, CONH2, CONHR, CONR2, CH2OH,.
NCO, NCOR, NHOH, NHNH2, NHNRH, CH2OCOR, CH2OCSR, COR, CSR, CSOR, CF3 et CCl3, et où R est alkyle, alkényle, aryle, aralkyle ou cycloalkyle;
où un sel pharmaceutiquement acceptable de celui-ci.
10. Peptide représenté par la formule générale I:
où
a est 1 et b est 0; ou b est 1 et a est 0; et d est 0-8; et
z est 1-7;
x est 1, y et q sont 1, et p est 0; ou
p est 1, x et q sont 0, et y est 1;
et en outre où,
si R
1 est H alors d est 0-8; ou
si R
1 n'est pas H, alors d est 0;
où R
1 est la chaîne latérale d'un acide amine sélectionné dans le groupe consistant en
alanine, arginine, asparagine, acide aspartique, cystéine, acide glutamique, glutamine,
glycine, histidine, isoleucine, leucine, lysine, méthionine, phénylalanine, proline,
sérine, thréonine, tryptophane, tyrosine et valine;
où R
2 est sélectionné dans le groupe consistant en NH
2,
NHR, NR
2, NR
3+H, OH, SH, RO, RS, RSO, RSO
2, CO
R, CSR, COOH, COOR, CONH
2, CONHR, CONR2, OCOR et SCOR, où R = alkyle, alkényle, aryle, aralkyle ou cycloalkyle;
où R
3 = H ou CH
3; et
où R
x est un cycle de carbone aromatique à 6 membres comprenant un substituant à la positon
4;
ou un sel pharmaceutiquement acceptable de celui-ci.
11. Peptide selon la revendication 10, où le cycle de carbone aromatique est substitué
par au moins un de: un alkyle inférieur, alkoxy, hydroxyle, carboxy, amine, thiol,
hydrazide, amide, halogénure, hydroxyl, éther, amine, nitrile, imine, nitro, sulfure,
sulfoxide, sulfane, thiol, aldéhyde, kéto, carboxy, ester, un groupe amide; un groupe
séléno, un groupe thio et leurs dérivés.
12. Peptide selon l'une quelconque des revendications précédentes, où le peptide comprend
un terminal N-libre, un terminal-C libre, ou à la fois un terminal N- et un terminal-C
libre.
13. Peptide selon l'une quelconque des revendications précédentes, où le sel pharmaceutiquement
acceptable est un sel d'addition d'acide, un sel métallique, un sel d'ammonium ou
un sel d'addition d'acide aminé.
14. Peptide selon la revendication 13, où le sel pharmaceutiquement acceptable est un
sel de chlorhydrate, un sel de sulfate, un sel de phosphate, un sel d'acétate, un
sel de maléate, un sel de fumarate, un sel de tartrate, un sel de citrate, un sel
de sodium, un sel de potassium, un sel de magnésium, un sel de calcium, un sel d'ammonium
ou de tétraméthyl d'ammonium, un sel de lysine, un sel de glycine ou un sel de phénylalanine.
15. Peptide selon l'une quelconque des revendications précédentes, où le peptide comprend
en outre un groupe de liaison d'hydrogène, et la distance entre le centre de masse
du groupe de liaison d'hydrogène et le groupe hydrophobe comprend entre environ 4
Angstrøms à environ 12 Angstrøms.
16. Peptide selon l'une quelconque des revendications précédentes, où le peptide comprend
en outre un groupe de liaison d'hydrogène, et la distance entre le centre de masse
du groupe de liaison d'hydrogène et le groupe hydrophobe comprend d'environ 5 Angstrøms
à environ 10 Angstrøms.
17. Peptide selon la revendication 10, où le substituant a un rayon de 3 à 11 Angstrøms.
18. Peptide selon la revendication 17, où le substituant est sélectionné dans le groupe
consistant en un groupe méthyle, éthyle, t-butyle, c-hexyle, phényle, n-butyle, n-hexyle,
n-octyle, éthoxy, t-butoxy, phénoxy, butoxy, benzyloxy, n-hexyloxy et un groupe n-octyloxy.
19. Peptide selon l'une quelconque des revendications précédentes, où le peptide fonctionne
comme médicament antiarythmique.
20. Peptide selon l'une quelconque des revendications précédentes, où le peptide est un
peptide disponible oralement.
21. Peptide selon l'une quelconque des revendications précédentes, où le peptide se lie
à un transporteur de hPepT1 ou un fragment biologiquement actif de celui-ci.
22. Peptide selon l'une quelconque des revendications précédentes, où le peptide a une
demi-vie dans un essai de stabilité de plasma in vitro de plus de 30 minutes.
23. Peptide selon l'une quelconque des revendications précédentes, où le peptide a une
demi-vie dans un essai de stabilité de plasma in vitro de plus qu'environ 48 heures.
24. Peptide selon l'une quelconque des revendications précédentes, où le peptide comprend
une liaison de peptide qui est modifiée pour stabiliser le peptide à l'encontre d'une
dégradation enzymatique.
25. Peptide selon l'une quelconque des revendications précédentes, où le peptide se lie
à un tissu, cellule ou fraction de cellule qui est un site d'action pour un peptide
antiarythmique.
26. Peptide selon la revendication 23, où le peptide antiarythmique est sélectionné dans
le groupe consistant en AAP, AAP10, HP5 ou un analogue fonctionnel de ceux-ci.
27. Peptide selon la revendication 23, où le peptide est un modulateur de la fonction
du tissu, de la cellule ou de la fraction de cellule.
28. Peptide selon la revendication 25, où le peptide antagonise la fonction du peptide
antiarythmique.
29. Peptide selon la revendication 25, où le peptide agonise la fonction de l'antiarythmique.
30. Peptide selon la revendication 23, où le peptide est un modulateur d'un récepteur
du peptide antiarythmique.
31. Peptide selon l'une quelconque des revendications précédentes, où le peptide est sélectionné
dans le groupe consistant en peptides indiqués dans le Tableau 1.
32. Peptide selon l'une quelconque des revendications précédentes, où le peptide est:
H-Gly-Lys(4-nitrobenzoyl)-OH (Composé 1);
H-Gly-Lys(4-méthoxybenzoyl)-OH (Composé 4);
H-D-Lys(4-méthoxybenzoyl)-Gly-OH (Composé 21);
H-D-Lys(4-nitrobenzoyl)Gly-OH (Composé 22);
H-D-Lys(4-t-butylbenzoyl)-Gly-OH (Composé 54);
H-D-Asn(NH(4-nitrobenzyl)Ala-OH (Composé 96);
H-D-Lys(benzoyl)Gly-OH (Composé 23) ou
H-D-Asn(NH(4-methoxybenzyl)Ala-OH (Composé 95).
33. Utilisation d'un peptide selon l'une quelconque des revendications 1 à 32 dans la
fabrication d'un médicament pour le traitement d'un état pathologique sélectionné
parmi une maladie cardiovasculaire, l'inflammation de l'épithélium des voies respiratoires,
une maladie du tissu alvéolaire, l'incontinence de la vessie, altération auditive,
une lésion endothéliale, la rétinopathie diabétique, la neuropathie diabétique, l'ischémie
du système nerveux central, l'ischémie de la moelle épinière, un trouble du tissu
dentaire, une maladie des reins, un échec d'une transplantation de moelle osseuse,
plaie, troubles d'érection, incontinence de vessie urinaire, douleur neuropathique,
inflammation sous-chronique et chronique, cancer, échec de transplantation, ostéoporose
ou une pathologie affectant la formation, croissance ou conservation de l'os.
34. Utilisation selon la revendication 33, où la maladie cardiovasculaire est sélectionnée
parmi l'arythmie, la cardiopathie ischémique aiguë, l'insuffisance cardiaque congestive,
les maladies de coeur congénitales, coeur pulmonaire, cardiomyopathie, myocardite
ou cardiopathie due à l'hypertension artérielle.
35. Utilisation selon la revendication 33 ou la revendication 34, où le médicament est
pour l'administration orale.
36. Utilisation selon l'une quelconque des revendications 33 à 35, où le médicament est
pour l'administration à un patient humain.